Atlantic Salmon (Salmo salar L.) as a Marine Functional Source of Gamma-Tocopherol

Gamma tocopherol (gT) exhibits beneficial cardiovascular effects partly due to its anti-inflammatory activity. Important sources of gT are vegetable oils. However, little is known to what extent gT can be transferred into marine animal species such as Atlantic salmon by feeding. Therefore, in this study we have investigated the transfer of dietary gT into salmon. To this end, fish were fed a diet supplemented with 170 ppm gT for 16 weeks whereby alpha tocopherol levels were adjusted to 190 ppm in this and the control diet. Feeding gT-rich diets resulted in a three-fold increase in gT concentrations in the liver and fillet compared to non-gT-supplemented controls. Tissue alpha tocopherol levels were not decreased indicating no antagonistic interaction between gamma- and alpha tocopherol in salmon. The concentration of total omega 3 fatty acids slightly increased in response to dietary gT. Furthermore, dietary gT significantly decreased malondialdehyde in the fillet, determined as a biomarker of lipid peroxidation. In the liver of gT fed salmon we observed an overall down-regulation of genes involved in lipid homeostasis. Additionally, gT improved the antioxidant capacity by up-regulating Gpx4a gene expression in the pyloric caeca. We suggest that Atlantic salmon may provide a marine functional source capable of enriching gT for human consumption.

Targeting a SWI/SNF-related chromatin remodeling complex to the β-globin promoter in erythroid cells

Chromatin remodeling complexes such as the SWI/SNF complex make DNA accessible to transcription factors by disrupting nucleosomes. However, it is not known how such complexes are targeted to the promoter. For example, a SWI/SNF1-like chromatin remodeling complex erythroid Krüppel-like factor (EKLF) coactivator-remodeling complex 1 (E-RC1) disrupts the nucleosomes over the human β-globin promoter in an EKLF-dependent manner. However, it is not known whether E-RC1 is targeted specifically to the β-globin promoter or whether E-RC1 is randomly targeted, but its activity is evident only at the β-globin promoter. Because E-RC1 cannot remodel chromatin over the β-globin promoter without EKLF in vitro, it has been proposed that SWI/SNF1-like complexes such as E-RC1 are targeted specifically to the promoter by selectively interacting with promoter-associated transcription factors such as EKLF. In this report, we test this hypothesis in the cellular context by using the ProteIN POsition Identification with Nuclease Tail (PIN*POINT) assay. We find that the Brahma-related gene (BRG) 1 and BRG1-associated factor (BAF) 170 subunits of E-RC1 are both recruited near the transcription initiation site of the β-globin promoter. On transiently transfected templates, both the locus control region and the EKLF-binding site are important for their recruitment to the β-globin promoter in mouse erythroleukemia cells. When the β-globin promoter was linked to the cytomegalovirus enhancer, the E-RC1 complex was not recruited, suggesting that recruitment of the E-RC1 complex is not a general property of enhancers.

The mouse histone H2a gene contains a small element that facilitates cytoplasmic accumulation of intronless gene transcripts and of unspliced HIV-1-related mRNAs

Histone mRNAs are naturally intronless and accumulate efficiently in the cytoplasm. To learn whether there are cis-acting sequences within histone genes that allow efficient cytoplasmic accumulation of RNAs, we made recombinant constructs in which sequences from the mouse H2a gene were cloned into a human β-globin cDNA. By using transient transfection and RNase protection analysis, we demonstrate here that a 100-bp sequence within the H2a coding region permits efficient cytoplasmic accumulation of the globin cDNA transcripts. We also show that this sequence appears to suppress splicing and can functionally replace Rev and the Rev-responsive element in the cytoplasmic accumulation of unspliced HIV-1-related mRNAs. Like the Rev-responsive element, this sequence acts in an orientation-dependent manner. We thus propose that the sequence identified here may be a member of the cis-acting elements that facilitate the cytoplasmic accumulation of naturally intronless gene transcripts.

Trichostatin A reverses skewed expression of CD154, interleukin-10, and interferon-γ gene and protein expression in lupus T cells

In systemic lupus erythematosus (SLE), T helper cells exhibit increased and prolonged expression of cell-surface CD40 ligand (CD154), spontaneously overproduce interleukin-10 (IL-10), but underproduce interferon-gamma (IFN-γ). We tested the hypothesis that the imbalance of these gene products reflects skewed expression of CD154, IL-10, and IFN-γ genes. Here, we demonstrate that the histone deacetylase inhibitor, trichostatin A, significantly down-regulated CD154 and IL-10 and up-regulated IFN-γ gene expression in SLE T cells. This reversal corrected the aberrant expression of these gene products, thereby enhancing IFN-γ production and inhibiting IL-10 and CD154 expression. That trichostatin A can simultaneously reverse the skewed expression of multiple genes implicated in the immunopathogenesis of SLE suggests that this pharmacologic agent may be a candidate for the treatment of this autoimmune disease.

Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation

Gamma oscillations synchronized between distant neuronal populations may be critical for binding together brain regions devoted to common processing tasks. Network modeling predicts that such synchrony depends in part on the fast time course of excitatory postsynaptic potentials (EPSPs) in interneurons, and that even moderate slowing of this time course will disrupt synchrony. We generated mice with slowed interneuron EPSPs by gene targeting, in which the gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was altered to drive expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunit GluR-B. GluR-B is a determinant of the relatively slow EPSPs in excitatory neurons and is normally expressed at low levels in γ-aminobutyric acid (GABA)ergic interneurons, but at high levels in the GAD-GluR-B mice. In both wild-type and GAD-GluR-B mice, tetanic stimuli evoked gamma oscillations that were indistinguishable in local field potential recordings. Remarkably, however, oscillation synchrony between spatially separated sites was severely disrupted in the mutant, in association with changes in interneuron firing patterns. The congruence between mouse and model suggests that the rapid time course of AMPA receptor-mediated EPSPs in interneurons might serve to allow gamma oscillations to synchronize over distance.

Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.

Conventional major histocompatibility complex (MHC) class I genes encode molecules that present intracellular peptide antigens to T cells. They are ubiquitously expressed and regulated by interferon gamma. Two highly divergent human MHC class I genes, MICA and MICB, are regulated by promoter heat shock elements similar to those of HSP70 genes. MICA encodes a cell surface glycoprotein, which is not associated with beta 2-microglobulin, is conformationally stable independent of conventional class I peptide ligands, and almost exclusively expressed in gastrointestinal epithelium. Thus, this MHC class I molecule may function as an indicator of cell stress and may be recognized by a subset of gut mucosal T cells in an unusual interaction.

Chromatin structure and gene expression.

It is now well understood that chromatin structure is perturbed in the neighborhood of expressed genes. This is most obvious in the neighborhood of promoters and enhancers, where hypersensitivity to nucleases marks sites that no longer carry canonical nucleosomes, and to which transcription factors bind. To study the relationship between transcription factor binding and the generation of these hypersensitive regions, we mutated individual cis-acting regulatory elements within the enhancer that lies between the chicken beta- and epsilon-globin genes. Constructions carrying the mutant enhancer were introduced by stable transformation into an avian erythroid cell line. We observed that weakening the enhancer resulted in creation of two classes of site: those still completely accessible to nuclease attack and those that were completely blocked. This all-or-none behavior suggests a mechanism by which chromatin structure can act to sharpen the response of developmental systems to changing concentrations of regulatory factors. Another problem raised by chromatin structure concerns the establishment of boundaries between active and inactive chromatin domains. We have identified a DNA element at the 5' end of the chicken beta-globin locus, near such a boundary, that has the properties of an insulator; in test constructions, it blocks the action of an enhancer on a promoter when it is placed between them. We describe the properties and partial dissection of this sequence. A third problem is posed by the continued presence of nucleosomes on transcribed genes, which might prevent the passage of RNA polymerase. We show, however, that a prokaryotic polymerase can transcribe through a histone octamer on a simple chromatin template. The analysis of this process reveals that an octamer is capable of transferring from a position in front of the polymerase to one behind, without ever losing its attachment to the DNA.

Disruption of epithelial gamma delta T cell repertoires by mutation of the Syk tyrosine kinase.

Chimeric mice in which lymphocytes are deficient in the Syk tyrosine kinase have been created. Compared with Syk-positive controls, mice with Syk -/- lymphocytes display substantial depletion of intraepithelial gamma delta T cells in the skin and gut, with developmental arrest occurring after antigen receptor gene rearrangement. In this dependence on Syk, subsets of intraepithelial gamma delta T cells are similar to B cells, but distinct from splenic gamma delta T cells that develop and expand in Syk-deficient mice. The characteristic associations of certain T-cell receptor V gamma/V delta gene rearrangements with specific epithelia are also disrupted by Syk deficiency.

RXR gamma null mice are apparently normal and compound RXR alpha +/-/RXR beta -/-/RXR gamma -/- mutant mice are viable.

The RXR gamma (RXR, retinoid X receptor) gene was disrupted in the mouse. Homozygous mutant mice developed normally and were indistinguishable from their RXR gamma +/- or wild-type littermates with respect to growth, fertility, viability, and apparent behavior in the animal facility. Moreover, RXR alpha -/-/RXR gamma -/- and RXR beta -/-/RXR gamma -/- mutant phenotypes were indistinguishable from those of RXR alpha -/- and RXR beta -/- mutants, respectively. Strikingly, RXR alpha +/-/RXR beta -/-/RXR gamma -/- triple mutants were viable. Thus, it appears that RXR gamma does not exert any essential function that cannot be performed by RXR alpha or RXR beta, and one copy of RXR alpha is sufficient to perform most of the functions of the RXRs.