Adapting Reach Up and Learn in Crisis and Conflict Settings: An Exploratory Multiple Case Study.

Objectives: In 2019, >71 million children aged <5 had spent their entire lives in conflict-affected settings. Compounding adversities including violence, poverty, and displacement have immediate and long-term effects on early childhood development, health, behavior, and well-being. In response, adaptations of Reach Up have been implemented in conflict and crisis settings.

Molecular basis for the dual subcellular distribution of microsomal glutathione transferase 1.

Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution.

Expression and Function of mARC: Roles in Lipogenesis and Metabolic Activation of Ximelagatran.

Recently two novel enzymes were identified in the outer mitochondrial membrane, mARC1 and mARC2. These molybdenum containing enzymes can reduce a variety of N-hydroxylated compounds, such as N-hydroxy-guanidines and sulfohydroxamic acids, as well as convert nitrite into nitric oxide (NO). However, their endogenous functions remain unknown.

An integrated in vitro model for simultaneous assessment of drug uptake, metabolism, and efflux.

The absorption, distribution, metabolism, and excretion (ADME) of drugs in vivo are to a large extent dependent on different transport and metabolism routes. Elucidation of this complex transport-metabolism interplay is a major challenge in drug development and at present no in vitro models suitable for this purpose are at hand. The aim of this study was to develop flexible, well-controlled, easy-to-use, integrated cell models, where drug transport and drug metabolism processes could be studied simultaneously.

Induced CYP3A4 expression in confluent Huh7 hepatoma cells as a result of decreased cell proliferation and subsequent pregnane X receptor activation.

We have previously shown that confluent growth of the human hepatoma cell line Huh7 substantially induces the CYP3A4 mRNA, protein, and activity levels. Here, the mechanisms behind were investigated, and a transcriptome analysis revealed significant up-regulation of liver-specific functions, whereas pathways related to proliferation and cell cycle were down-regulated in the confluent cells. Reporter analysis revealed that the CYP3A4 gene was transcriptionally activated during confluence in a process involving pregnane X receptor (PXR).

Amidoxime reductase system containing cytochrome b5 type B (CYB5B) and MOSC2 is of importance for lipid synthesis in adipocyte mitochondria.

Reduction of hydroxylamines and amidoximes is important for drug activation and detoxification of aromatic and heterocyclic amines. Such a reductase system was previously found to be of high activity in adipose tissue and liver, and furthermore, in vitro studies using recombinant truncated and purified enzymes suggested the participation of cytochrome b(5) reductase (CYB5R), cytochrome b(5) (CYB5), and molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and -2). Here, we show that purified rat liver outer mitochondrial membrane contains high amidoxime reductase activity and that MOSC2 is exclusively localized to these membranes.

IL-4-mediated transcriptional regulation of human CYP2E1 by two independent signaling pathways.

Cytochrome P450 2E1 (CYP2E1), the alcohol-inducible member of the cytochrome P450 super family, plays an important role in both physiological and pathophysiological processes. The present study focused on the induction of human CYP2E1 transcription by the anti-inflammatory cytokine interleukin-4 (IL-4) in human hepatoma B16A2 cells and revealed that this regulation is mediated by two independent pathways. RNA interference and overexpression of STAT6, indicated that the JAK-STAT signaling pathway is involved in IL-4-dependent induction and mutagenesis revealed the presence of a STAT6 binding site in CYP2E1 proximal promoter region (-583/-574-bp).

CYP3A4 catalytic activity is induced in confluent Huh7 hepatoma cells.

Drug-induced hepatotoxicity is an important cause for disapproval, limitations of use, or withdrawal of drugs, and there is a high need for reproducible in vitro systems that can predict such toxicity. In this study, we show that confluent growth of the human hepatoma cell line Huh7 up to 5 weeks results in increased gene expression of several cytochromes P450 (P450s), UDP-glucuronosyltransferases, transporters, transcription factors, and several liver-specific genes, as measured by low-density array. The most striking effect was seen for CYP3A4 expression.

Cytochrome P450 proteins: retention and distribution from the endoplasmic reticulum.

Cytochrome P450 (CYP) is a large family of well-conserved integral membrane proteins localized primarily in the membrane of the endoplasmic reticulum (ER), where these enzymes metabolize a variety of both endogenous and exogenous compounds. It has become apparent that these microsomal CYP proteins are also present in other cellular compartments, such as the cell surface and in mitochondria, where the enzymes display catalytic activity toward CYP-specific substrates, in some cases with altered substrate specificity. CYP-drug adducts exposed at the cell surface are important mediators of idiosyncratic drug toxicities.

A SNAIL1-SMAD3/4 transcriptional repressor complex promotes TGF-beta mediated epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is essential for organogenesis and is triggered during carcinoma progression to an invasive state. Transforming growth factor-beta (TGF-beta) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT.

Intracellular transport and localization of microsomal cytochrome P450.

The cytochrome P450 (P450) enzymes are mainly localized to the endoplasmic reticulum (ER), where they function within catalytic complexes metabolizing xenobiotics and some endogenous substrates. However, certain members of families 1-3 were also found in other subcellular compartments, such as mitochondria, plasma membrane, and lysosomes. The physiological function of these enzymes in non-ER locations is not known, although plasma-membrane-associated P450s have been described to be catalytically active and to participate in immune-mediated reactions with autoantibody formation that can trigger drug-induced hepatitis.

The coxsackie and adenovirus receptor (CAR) is required for renal epithelial differentiation within the zebrafish pronephros.

The coxsackie and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily and a component of vertebrate tight junctions. CAR protein is widely expressed in fish and mammals in organs of epithelial origin suggesting possible functions in epithelial biology. In order to gain insight into its function, we knocked the CAR gene down in zebrafish using antisense morpholinos.

The cytoplasmic tails of Uukuniemi Virus (Bunyaviridae) G(N) and G(C) glycoproteins are important for intracellular targeting and the budding of virus-like particles.

Functional motifs within the cytoplasmic tails of the two glycoproteins G(N) and G(C) of Uukuniemi virus (UUK) (Bunyaviridae family) were identified with the help of our recently developed virus-like particle (VLP) system for UUK virus (A. K. Overby, V.

Exosomes with immune modulatory features are present in human breast milk.

Breast milk is a complex liquid with immune-competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant's immune system. Exosomes are nanovesicles (30-100 nm) with an endosome-derived limiting membrane secreted by a diverse range of cell types. Because exosomes carry immunorelevant structures, they are suggested to participate in directing the immune response.

The glycoprotein cytoplasmic tail of Uukuniemi virus (Bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging.

We have analyzed the importance of specific amino acids in the cytoplasmic tail of the glycoprotein G(N) for packaging of ribonucleoproteins (RNPs) into virus-like particles (VLPs) of Uukuniemi virus (UUK virus), a member of the Bunyaviridae family. In order to study packaging, we added the G(N)/G(C) glycoprotein precursor (p110) to a polymerase I-driven minigenome rescue system to generate VLPs that are released into the supernatant. These particles can infect new cells, and reporter gene expression can be detected.

Generation and analysis of infectious virus-like particles of uukuniemi virus (bunyaviridae): a useful system for studying bunyaviral packaging and budding.

In the present report we describe an infectious virus-like particle (VLP) system for the Uukuniemi (UUK) virus, a member of the Bunyaviridae family. It utilizes our recently developed reverse genetic system based on the RNA polymerase I minigenome system for UUK virus used to study replication, encapsidation, and transcription by monitoring reporter gene expression. Here, we have added the glycoprotein precursor expression plasmid together with the minigenome, nucleoprotein, and polymerase to generate VLPs, which incorporate the minigenome and are released into the supernatant.

Oligomerization and interacellular localization of the glycoprotein receptor ERGIC-53 is independent of disulfide bonds.

ERGIC-53 is a type I transmembrane lectin facilitating the efficient export of a subset of secretory glycoproteins from the endoplasmic reticulum. Previous results have shown that ERGIC-53 is present as reduction-sensitive homo-oligomers, i.e.

Four permeases import proline and the toxic proline analogue azetidine-2-carboxylate into yeast.

We have found that proline and the toxic proline analogue azetidine-2-carboxylate (AzC) are efficiently imported into Saccharomyces cerevisiae cells by four amino acid permeases, including two nitrogen-regulated permeases (PUT4 and GAP1) and two permeases that are regulated by the SPS sensor of extracellular amino acids (AGP1 and GNP1). In contrast to Agp1p, Gnp1p is not functionally expressed when cells are grown on media containing proline as sole nitrogen source. These findings have implications for the interpretation of studies using AzC to characterize nitrogen source-dependent regulation of amino acid uptake and of post-Golgi targeting and localization of amino acid permeases in yeast.

Identification of sequences responsible for intracellular targeting and membrane binding of rat CYP2E1 in yeast.

The role of the hydrophobic NH(2)-terminal domain of rat CYP2E1 for intracellular targeting and membrane binding was investigated in Saccharomyces cerevisiae as a model system. Several different CYP2E1 variants with deletions and mutations were expressed in yeast, and their intracellular localization and membrane-binding properties were analyzed. We found that an amino acid stretch including the B-helix from glycine 82 to asparagine 95 is responsible for mitochondrial association of CYP2E1 in yeast.

VIPL, a VIP36-like membrane protein with a putative function in the export of glycoproteins from the endoplasmic reticulum.

Subsets of glycoproteins are thought to require lectin-like membrane receptors for efficient export out of the endoplasmic reticulum (ER). To identify new members related to two previously characterized intracellular lectins ERGIC-53/p58 and VIP36, we carried out an extensive database search using the conserved carbohydrate recognition domain (CRD) as a search string. A gene, more closely related to VIP36 than to ERGIC-53/p58, and hence called VIPL (VIP36-Like), was identified.

Ethanol and oxidative stress.

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chair was Albert Y. Sun.

Identification and tissue distribution of the novel human cytochrome P450 2S1 (CYP2S1).

With the aid of the htgs and dbEST databases, a novel cytochrome P450 cDNA was found by homology searches, and the corresponding gene was identified on chromosome 19. Nested PCR was used to amplify a full-length sequence of 1515 bp. The predicted 504 amino acid sequence displays 38--49% identity with CYP2 family members and the protein was designated CYP2S1.