Timing the Generation of Distinct Retinal Cells by Homeobox Proteins

The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). Furthermore, by in vivo lipofection of “sensors” in which green fluorescent protein translation is under control of the 3′ untranslated region (UTR), we found that the 3′ UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities

A specific box switches the cell fate determining activity of XOTX2 and XOTX5b in the Xenopus retina

<p>Abstract</p> <p>Background</p> <p><it>Otx </it>genes, orthologues of the <it>Drosophila orthodenticle </it>gene (<it>otd</it>), play crucial roles in vertebrate brain development. In the <it>Xenopus </it>eye, <it>Xotx2 </it>and <it>Xotx5b </it>promote bipolar and photoreceptor cell fates, respectively. The molecular basis of their differential action is not completely understood, though the carboxyl termini of the two proteins seem to be crucial. To define the molecular domains that make the action of these proteins so different, and to determine whether their retinal abilities are shared by <it>Drosophila </it>OTD, we performed an <it>in vivo </it>molecular dissection of their activity by transfecting retinal progenitors with several wild-type, deletion and chimeric constructs of <it>Xotx2</it>, <it>Xotx5b </it>and <it>otd</it>.</p> <p>Results</p> <p>We identified a small 8–10 amino acid divergent region, directly downstream of the homeodomain, that is crucial for the respective activities of XOTX2 and XOTX5b. In lipofection experiments, the exchange of this 'specificity box' completely switches the retinal activity of XOTX5b into that of XOTX2 and <it>vice versa</it>. Moreover, the insertion of this box into <it>Drosophila </it>OTD, which has no effect on retinal cell fate, endows it with the specific activity of either XOTX protein. Significantly, in cell transfection experiments, the diverse ability of XOTX2 and XOTX5b to synergize with NRL, a cofactor essential for vertebrate rod development, to transactivate the rhodopsin promoter is also switched depending on the box. We also show by GST-pull down that XOTX2 and XOTX5b differentially interact with NRL, though this property is not strictly dependent on the box.</p> <p>Conclusion</p> <p>Our data provide molecular evidence on how closely related homeodomain gene products can differentiate their functions to regulate distinct cell fates. A small 'specificity box' is both necessary and sufficient to confer on XOTX2 and XOTX5b their distinct activities in the developing frog retina and to convert the neutral orthologous OTD protein of <it>Drosophila </it>into a positive and specific XOTX-like retinal regulator. Relatively little is known of what gives developmental specificity to homeodomain regulators. We propose that this box is a major domain of XOTX proteins that provides them with the appropriate developmental specificity in retinal histogenesis.</p

Interaction of an atypical Plasmodium falciparum ETRAMP with human apolipoproteins

Background: In order to establish a successful infection in the human host, the malaria parasite Plasmodium falciparum must establish interactions with a variety of human proteins on the surface of different cell types, as well as with proteins inside the host cells. To better understand this aspect of malaria pathogenesis, a study was conducted with the goal of identifying interactions between proteins of the parasite and those of its human host. Methods: A modified yeast two-hybrid methodology that preferentially selects protein fragments that can be expressed in yeast was used to conduct high-throughput screens with P. falciparum protein fragments against human liver and cerebellum libraries. The resulting dataset was analyzed to exclude interactions that are not likely to occur in the human host during infection. Results: An initial set of 2,200 interactions was curated to remove proteins that are unlikely to play a role in pathogenesis based on their annotation or localization, and proteins that behave promiscuously in the two-hybrid assay, resulting in a final dataset of 456 interactions. A cluster that implicates binding between P. falciparum PFE1590w/ETRAMP5, a putative parasitophorous vacuole membrane protein, and human apolipoproteins ApoA, ApoB and ApoE was selected for further analysis. Different isoforms of ApoE, which are associated with different outcomes of malaria infection, were shown to display differential interactions with PFE1590w. Conclusion: A dataset of interactions between proteins of P. falciparum and those of its human host was generated. The preferential interaction of the P. falciparum PFE1590w protein with the human ApoE e3 and ApoE e4 isoforms, but not the ApoE e2 isoform, supports the hypothesis that ApoE genotype affects risk of malaria infection. The dataset contains other interactions of potential relevance to disease that may identify possible vaccine candidates and drug targets.This work was supported in part by grant P50 GM64655 from the NIH

Probing the Origins of the CIV and Fe Kalpha Baldwin Effect

We use UV/optical and X-ray observations of 272 radio-quiet Type 1 AGNs and quasars to investigate the CIV Baldwin Effect (BEff). The UV/optical spectra are drawn from the Hubble Space Telescope, International Ultraviolet Explorer and Sloan Digital Sky Survey archives. The X-ray spectra are from the Chandra and XMM-Newton archives. We apply correlation and partial-correlation analyses to the equivalent widths, continuum monochromatic luminosities, and alpha_ox, which characterizes the relative X-ray to UV brightness. The equivalent width of the CIV 1549 emission line is correlated with both alpha_ox and luminosity. We find that by regressing l_UV with EW(CIV) and alpha_ox, we can obtain tighter correlations than by regressing l_UV with only EW(CIV). Both correlation and regression analyses imply that l_UV is not the only factor controlling the changes of EW(CIV); alpha_ox (or, equivalently, the soft X-ray emission) plays a fundamental role in the formation and variation of CIV. Variability contributes at least 60% of the scatter of the EW(CIV)-l_UV relation and at least 75% of the scatter of the of the EW(CIV)-alpha_ox relation. In our sample, narrow Fe Kalpha 6.4 keV emission lines are detected in 50 objects. Although narrow Fe Kalpha exhibits a BEff similar to that of CIV, its equivalent width has almost no dependence on either alpha_ox or EW(CIV). This suggests that the majority of narrow Fe Kalpha emission is unlikely to be produced in the broad emission-line region. We do find suggestive correlations between the emission-line luminosities of CIV and Fe Kalpha, which could be potentially used to estimate the detectability of the Fe Kalpha line of quasars from rest-frame UV spectroscopic observations

The X-Ray Properties of the Optically Brightest Mini-BAL Quasars from the Sloan Digital Sky Survey

We have compiled a sample of 14 of the optically brightest radio-quiet quasars ($m_{i}$~$\le$~17.5 and $z$~$\ge$~1.9) in the Sloan Digital Sky Survey Data Release 5 quasar catalog that have C IV mini-BALs present in their spectra. X-ray data for 12 of the objects were obtained via a Chandra snapshot survey using ACIS-S, while data for the other two quasars were obtained from archival XMM-Newton observations. Joint X-ray spectral analysis shows the mini-BAL quasars have a similar average power-law photon index ($\Gamma\approx1.9$) and level of intrinsic absorption ($N_H \lesssim 8\times 10^{21} \ {\rm cm}^{-2}$) as non-BMB (neither BAL nor mini-BAL) quasars. Mini-BAL quasars are more similar to non-BMB quasars than to BAL quasars in their distribution of relative X-ray brightness (assessed with $\Delta\alpha_{\rm ox}$). Relative colors indicate mild dust reddening in the optical spectra of mini-BAL quasars. Significant correlations between $\Delta\alpha_{\rm ox}$ and UV absorption properties are confirmed for a sample of 56 sources combining mini-BAL and BAL quasars with high signal-to-noise ratio rest-frame UV spectra, which generally supports models in which X-ray absorption is important in enabling driving of the UV absorption-line wind. We also propose alternative parametrizations of the UV absorption properties of mini-BAL and BAL quasars, which may better describe the broad absorption troughs in some respects.Comment: ApJ accepted; 21 pages, 11 figures, and 9 table

Cell-specific pattern of berberine pleiotropic effects on different human cell lines

The natural alkaloid berberine has several pharmacological properties and recently received attention as a potential anticancer agent. In this work, we investigated the molecular mechanisms underlying the anti-Tumor effect of berberine on glioblastoma U343 and pancreatic carcinoma MIA PaCa-2 cells. Human dermal fibroblasts (HDF) were used as non-cancer cells. We show that berberine differentially affects cell viability, displaying a higher cytotoxicity on the two cancer cell lines than on HDF. Berberine also affects cell cycle progression, senescence, caspase-3 activity, autophagy and migration in a cell-specific manner. In particular, in HDF it induces cell cycle arrest in G2 and senescence, but not autophagy; in the U343 cells, berberine leads to cell cycle arrest in G2 and induces both senescence and autophagy; in MIA PaCa-2 cells, the alkaloid induces arrest in G1, senescence, autophagy, it increases caspase-3 activity and impairs migration/invasion. As demonstrated by decreased citrate synthase activity, the three cell lines show mitochondrial dysfunction following berberine exposure. Finally, we observed that berberine modulates the expression profile of genes involved in different pathways of tumorigenesis in a cell line-specific manner. These findings have valuable implications for understanding the complex functional interactions between berberine and specific cell types

Protopine/Gemcitabine Combination Induces Cytotoxic or Cytoprotective Effects in Cell Type-Specific and Dose-Dependent Manner on Human Cancer and Normal Cells

The natural alkaloid protopine (PRO) exhibits pharmacological properties including anticancer activity. We investigated the effects of PRO, alone and in combination with the chemotherapeutic gemcitabine (GEM), on human tumor cell lines and non-tumor human dermal fibroblasts (HDFs). We found that treatments with different PRO/GEM combinations were cytotoxic or cytoprotective, depending on concentration and cell type. PRO/GEM decreased viability in pancreatic cancer MIA PaCa-2 and PANC-1 cells, while it rescued the GEM-induced viability decline in HDFs and in tumor MCF-7 cells. Moreover, PRO/GEM decreased G1, S and G2/M phases, concomitantly with an increase of subG1 phase in MIA PaCa-2 and PANC-1 cells. Differently, PRO/GEM restored the normal progression of the cell cycle, altered by GEM, and decreased cell death in HDFs. PRO alone increased mitochondrial reactive oxygen species (ROS) in MIA PaCa-2, PANC-1 cells and HDFs, while PRO/GEM increased both intracellular and mitochondrial ROS in the three cell lines. These results indicate that specific combinations of PRO/GEM may be used to induce cytotoxic effects in pancreatic tumor MIA PaCa-2 and PANC-1 cells, but have cytoprotective or no effects in HDFs

Variable X-ray Absorption in the Seyfert 2 Galaxy Mrk 348

We present RXTE monitoring observations of the Seyfert 2 galaxy Mrk 348 spanning a 6 month period. The time-averaged spectrum in the 3-20 keV band shows many features characteristic of a Compton-thin Seyfert 2 galaxy, namely a hard underlying power-law continuum (photon index = 1.8) with heavy soft X-ray absorption (N_h ~ 10^23 cm^-2) plus measureable iron line emission (equivalent width ~ 100 eV) and, at high energy, evidence for a reflection component (R < 1). During the first half of the monitoring period the X-ray continuum flux from Mrk 348 remained relatively steady. However this was followed by a significant brightening of the source (by roughly a factor of 4) with the fastest change corresponding to a doubling of its X-ray flux on a timescale of about 20 days. The flux increase was accompanied by a marked softening of X-ray spectrum most likely attributable to a factor 3 decline in the intrinsic line-of-sight column density. In contrast the iron line and the reflection components showed no evidence of variability. These observations suggest a scenario in which the central X-ray source is surrounded by a patchy distribution of absorbing material located within about a light-week of the nucleus of Mrk 348. The random movement of individual clouds within the absorbing screen, across our line of sight, produces substantial temporal variations in the measured column density on timescales of weeks to months and gives rise to the observed X-ray spectral variability. However, as viewed from the nucleus the global coverage and typical thickness of the cloud layer remains relatively constant.Comment: 19 pages, 3 figures Accepted for publication in the Astrophysical Journa

A Search for H2O Megamasers in High-z Type-2 AGNs

We report a search for H2O megamasers in 274 SDSS type-2 AGNs (0.3 < z < 0.83), half of which can be classified as type-2 QSOs from their [OIII] 5007 luminosity, using the Robert C. Byrd Green Bank Telescope (GBT) and the Effelsberg 100-m radio telescope. Apart from the detection of the extremely luminous water vapor megamaser SDSS J080430.99+360718.1, already reported by Barvainis & Antonucci (2005), we do not find any additional line emission. This high rate of non-detections is compared to the water maser luminosity function created from the 78 water maser galaxies known to date and its extrapolation towards the higher luminosities of "gigamasers" that we would have been able to detect given the sensitivity of our survey. The properties of the known water masers are summarized and discussed with respect to the nature of high-z type-2 AGNs and megamasers in general. In the appendix, we list 173 additional objects (mainly radio galaxies, but also QSOs and galaxies) that were observed with the GBT, the Effelsberg 100-m radio telescope, or Arecibo Observatory without leading to the detection of water maser emission.Comment: 28 pages, 3 figures. Accepted for publication in the Astrophysical Journa

A eutherian-specific microRNA controls the translation of Satb2 in a model of cortical differentiation

Cerebral cortical development is controlled by key transcription factors that specify the neuronal identities in the different layers. The mechanisms controlling their expression in distinct cells are only partially known. We investigated the expression and stability of Tbr1, Bcl11b, Fezf2, Satb2, and Cux1 mRNAs in single developing mouse cortical cells. We observe that Satb2 mRNA appears much earlier than its protein and in a set of cells broader than expected, suggesting an initial inhibition of its translation, subsequently released during development. Mechanistically, Satb2 30UTR modulates protein translation of GFP reporters during mouse corticogenesis. We select miR541, a eutherian-specific miRNA, and miR-92a/b as the best candidates responsible for SATB2 inhibition, being strongly expressed in early and reduced in late progenitor cells. Their inactivation triggers robust and premature SATB2 translation in both mouse and human cortical cells. Our findings indicate RNA interference as a major mechanism in timing cortical cell identities