Redistribution of Transcription Factor AP-2α in Differentiating Cultured Human Epidermal Cells

Expression of the transcription factor AP-2α was examined in cultured human epidermal cells. Levels of AP-2α mRNA increased substantially after the cultures reached confluence, similar to the expression pattern of the differentiation markers involucrin and keratinocyte transglutaminase. The level of AP-2α protein in nuclear extracts declined markedly after confluence, however, along with its ability to form complexes with oligonucleotides containing the AP-2 response element. In contrast, the levels of AP-2α protein in cytoplasmic extracts increased dramatically after confluence, but these extracts had low DNA binding activity. Supershift experiments with specific antisera detected only AP-2α and not the β or γ isoforms. Examination of its localization by confocal microscopy revealed that AP-2α was primarily in the nucleus of basal cells and largely cytoplasmic in the most superficial cells. Localization was a dynamic phenomenon in that changing the medium resulted in accumulation of this transcription factor in the nucleus after several hours. Overall, the data indicate that AP-2α transcriptional activity is regulated in a differentiation-dependent manner in cultured keratinocytes and that this occurs by relocalization of the protein. Nuclear localization of the AP-2α protein in basal cells permits its accessibility to response elements in gene promoters, whereas sequestration in the cytoplasm as the differentiation program progresses curtails its transcriptional activity. This regulatory scheme may provide keratinocytes with the ability to restore AP-2 transcriptional activity rapidly by redistribution to the nucleus after receiving an appropriate growth signal, such as a medium change

Redistribution of Transcription Factor AP-2α in Differentiating Cultured Human Epidermal Cells

Expression of the transcription factor AP-2α was examined in cultured human epidermal cells. Levels of AP-2α mRNA increased substantially after the cultures reached confluence, similar to the expression pattern of the differentiation markers involucrin and keratinocyte transglutaminase. The level of AP-2α protein in nuclear extracts declined markedly after confluence, however, along with its ability to form complexes with oligonucleotides containing the AP-2 response element. In contrast, the levels of AP-2α protein in cytoplasmic extracts increased dramatically after confluence, but these extracts had low DNA binding activity. Supershift experiments with specific antisera detected only AP-2α and not the β or γ isoforms. Examination of its localization by confocal microscopy revealed that AP-2α was primarily in the nucleus of basal cells and largely cytoplasmic in the most superficial cells. Localization was a dynamic phenomenon in that changing the medium resulted in accumulation of this transcription factor in the nucleus after several hours. Overall, the data indicate that AP-2α transcriptional activity is regulated in a differentiation-dependent manner in cultured keratinocytes and that this occurs by relocalization of the protein. Nuclear localization of the AP-2α protein in basal cells permits its accessibility to response elements in gene promoters, whereas sequestration in the cytoplasm as the differentiation program progresses curtails its transcriptional activity. This regulatory scheme may provide keratinocytes with the ability to restore AP-2 transcriptional activity rapidly by redistribution to the nucleus after receiving an appropriate growth signal, such as a medium change

Potent Phototoxicity of Marine Bunker Oil to Translucent Herring Embryos after Prolonged Weathering

Pacific herring embryos (Clupea pallasi) spawned three months following the Cosco Busan bunker oil spill in San Francisco Bay showed high rates of late embryonic mortality in the intertidal zone at oiled sites. Dead embryos developed to the hatching stage (e.g. fully pigmented eyes) before suffering extensive tissue deterioration. In contrast, embryos incubated subtidally at oiled sites showed evidence of sublethal oil exposure (petroleum-induced cardiac toxicity) with very low rates of mortality. These field findings suggested an enhancement of oil toxicity through an interaction between oil and another environmental stressor in the intertidal zone, such as higher levels of sunlight-derived ultraviolet (UV) radiation. We tested this hypothesis by exposing herring embryos to both trace levels of weathered Cosco Busan bunker oil and sunlight, with and without protection from UV radiation. Cosco Busan oil and UV co-exposure were both necessary and sufficient to induce an acutely lethal necrotic syndrome in hatching stage embryos that closely mimicked the condition of dead embryos sampled from oiled sites. Tissue levels of known phototoxic polycyclic aromatic compounds were too low to explain the observed degree of phototoxicity, indicating the presence of other unidentified or unmeasured phototoxic compounds derived from bunker oil. These findings provide a parsimonious explanation for the unexpectedly high losses of intertidal herring spawn following the Cosco Busan spill. The chemical composition and associated toxicity of bunker oils should be more thoroughly evaluated to better understand and anticipate the ecological impacts of vessel-derived spills associated with an expanding global transportation network

Targets of the Entamoeba histolytica Transcription Factor URE3-BP

The Entamoeba histolytica transcription factor Upstream Regulatory Element 3-Binding Protein (URE3-BP) is a calcium-responsive regulator of two E. histolytica virulence genes, hgl5 and fdx1. URE3-BP was previously identified by a yeast one-hybrid screen of E. histolytica proteins capable of binding to the sequence TATTCTATT (Upstream Regulatory Element 3 (URE3)) in the promoter regions of hgl5 and fdx1. In this work, precise definition of the consensus URE3 element was performed by electrophoretic mobility shift assays (EMSA) using base-substituted oligonucleotides, and the consensus motif validated using episomal reporter constructs. Transcriptome profiling of a strain induced to produce a dominant-positive URE3-BP was then used to identify additional genes regulated by URE3-BP. Fifty modulated transcripts were identified, and of these the EMSA defined motif T[atg]T[tc][cg]T[at][tgc][tg] was found in over half of the promoters (54% p<0.0001). Fifteen of the URE3-BP regulated genes were potential membrane proteins, suggesting that one function of URE3-BP is to remodel the surface of E. histolytica in response to a calcium signal. Induction of URE3-BP leads to an increase in tranwell migration, suggesting a possible role in the regulation of cellular motility

New Frontiers-class Uranus Orbiter: Exploring the feasibility of achieving multidisciplinary science with a mid-scale mission

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Factors Affecting Sperm Motility, Fertilization and Early Development in the Pacific Herring (Clupea pallasi)

The effects of an environmental stressor (salinity) and an anthropogenic stressor (creosote-derived compounds) on sperm motility, fertilization and early development were investigated in the Pacific herring. In the laboratory, the optimal salinity for sperm motility initiation in response to an egg-derived factor, sperm motility initiating factor (SMIF), was 16-24 parts per thousand (ppt), while the optimal salinity for both fertilization and development was 12-24 ppt. These salinities are approximately one-half that of full-strength seawater. These laboratory data correlated with the optimal salinity for development (16-20 ppt) for embryos deployed at field sites in San Francisco Bay. The effect of individual cations on fertilization was also assessed. Sodium (Na·) inhibited fertilization at concentrations ≤55 or ≥440 mM. Alterations in calcium or magnesium had no effect on fertilization unless omitted from the medium. A similar effect was seen for potassium (K·), but elevated K. in combination with elevated Na· inhibited fertilization to a greater degree than Na· alone.To characterize the mechanism for the effects of altered salinity on sperm motility, changes in intracellular ions in response to SMIF or by manipulation of extracellular ions were investigated. SMIF induced a membrane depolarization that was inhibited by increasing either salinity or the Na' concentration. SMIF, and an activator of protein kinase C. induced an increase in intracellular Ca2+ ((Ca2+]i), which along with motility initiation required the presence of extracellular Ca2+ ((Ca2+]o). An efflux of Na· was observed in response to SMIF, or in the absence of SMIF, in low sodium media. again requiring [ca2+]o. Membrane depolarization. increase in [Ca2+]i, and Na· efflux were inhibited by calcium channel and sodium-calcium exchange inhibitors.Finally, the effects of creosote-derived compounds on development were investigated. Exposed embryos exhibited significant mortality, delay in development, cardiac abnormalities, abnormal movement within the chorion. and edema. Hatching success of exposed embryos was also decreased, and all larvae exhibited severe morphological deforinities (scoliosis, pericardia! and/or yolk-sac edema. Less than optimal salinities (8 and 28 ppt) enhanced the effects of creosote-derived compounds on development

Factors Affecting Sperm Motility, Fertilization and Early Development in the Pacific Herring (Clupea pallasi)

The effects of an environmental stressor (salinity) and an anthropogenic stressor (creosote-derived compounds) on sperm motility, fertilization and early development were investigated in the Pacific herring. In the laboratory, the optimal salinity for sperm motility initiation in response to an egg-derived factor, sperm motility initiating factor (SMIF), was 16-24 parts per thousand (ppt), while the optimal salinity for both fertilization and development was 12-24 ppt. These salinities are approximately one-half that of full-strength seawater. These laboratory data correlated with the optimal salinity for development (16-20 ppt) for embryos deployed at field sites in San Francisco Bay. The effect of individual cations on fertilization was also assessed. Sodium (Na·) inhibited fertilization at concentrations ≤55 or ≥440 mM. Alterations in calcium or magnesium had no effect on fertilization unless omitted from the medium. A similar effect was seen for potassium (K·), but elevated K. in combination with elevated Na· inhibited fertilization to a greater degree than Na· alone.To characterize the mechanism for the effects of altered salinity on sperm motility, changes in intracellular ions in response to SMIF or by manipulation of extracellular ions were investigated. SMIF induced a membrane depolarization that was inhibited by increasing either salinity or the Na' concentration. SMIF, and an activator of protein kinase C. induced an increase in intracellular Ca2+ ((Ca2+]i), which along with motility initiation required the presence of extracellular Ca2+ ((Ca2+]o). An efflux of Na· was observed in response to SMIF, or in the absence of SMIF, in low sodium media. again requiring [ca2+]o. Membrane depolarization. increase in [Ca2+]i, and Na· efflux were inhibited by calcium channel and sodium-calcium exchange inhibitors.Finally, the effects of creosote-derived compounds on development were investigated. Exposed embryos exhibited significant mortality, delay in development, cardiac abnormalities, abnormal movement within the chorion. and edema. Hatching success of exposed embryos was also decreased, and all larvae exhibited severe morphological deforinities (scoliosis, pericardia! and/or yolk-sac edema. Less than optimal salinities (8 and 28 ppt) enhanced the effects of creosote-derived compounds on development

Polycyclic aromatic hydrocarbons disrupt axial development in sea urchin embryos through a β-catenin dependent pathway

Sea urchin ( Lytechinus anemesis) embryos were used as an experimental system to investigate the mechanisms of the developmental toxicity of creosote, one of the most widely used wood preserving chemicals, as well as some of its polycyclic aromatic hydrocarbon (PAH) constituents (phenanthrene, fluoranthene, fluorene, pyrene and quinoline). Data suggest that creosote and PAHs affect axial development and patterning in sea urchin embryos by disrupting the regulation of β-catenin, a crucial transcriptional co-activator of specific target genes in the Wnt/wg signaling pathway. When ciliated blastula stage embryos were exposed to these compounds, they developed into exogastrulae with completely evaginated archentera, demonstrating that these chemicals disrupt axial development and patterning. This response occurred in a dose-dependent fashion, with the EC 50 of creosote for complete exogastrulation being 1.57 ppm, while the EC 50s of the PAHs ranged from 0.41 ppm (2.0 μM) to 4.33 ppm (33.5 μM). Morphologically, the exogastrulae that developed from embryos exposed to creosote and PAHs appeared to be identical to those that resulted from exposure to lithium chloride, a classical agent known to induce vegetalization and exogastrulation in sea urchin embryos. Immunological studies using antibodies against β-catenin, a multi-functional protein known to be involved in cell–cell adhesion and cell fate specification during embryonic development, revealed high levels of nuclear accumulation of β-catenin by cells of creosote- and PAH-exposed embryos, irrespective of their positions in the developing embryo. Dissociated embryonic cells cultured in the presence of these agents rapidly responded in a similar fashion. Since β-catenin accumulation occurs in nuclei of several types of cancer cells, it is possible this may be a general mechanism by which PAHs affect a variety of different cell types

Integrating contaminant responses in indicator saltmarsh species.

A challenge in environmental management is to provide both methodology and a framework for assessing effects of pollutants in resident species and then applying the findings to management. The Pacific Estuarine Ecosystem Indicator Research (PEEIR) consortium advocates the development of an integrated portfolio of techniques using indicator species selected for various habitat types. We developed such a portfolio for California salt marsh ecosystems and evaluated the feasibility of our approach in management applications. PEEIR is employing a suite of biomarker responses in two indigenous species, the lined shore crab (Pachygrapsus crassipes) and the longjaw mudsucker (Gillichthys mirabilis). Detrimental effects such as apoptosis, endocrine disruption, and ovarian tumors have been observed in G. mirabilis at a site where toxicity test responses were relatively low. With P. crassipes, developmental abnormalities and several markers of decreased reproductive performance were quantified at the same site. Multivariate statistical techniques are used to examine the relationships between the responses and multiple contaminant and natural stressors. For the fish, findings are related to population-level parameters using dynamic energy budget (DEB) models