Characterization of UEF-4, a DNA-binding Protein Required for Transcriptional Synergism between Two AP-1 Sites in the Human Urokinase Enhancer

Abstract The enhancer of the inducible urokinase gene depends on three essential but not sufficient transactivating elements, an upstream PEA3/AP-1A and a downstream AP-1B site. Enhancer activity also requires the interposed 74-base pair-long cooperation mediator (COM) region that allows transcriptional synergism between the transactivating sites. The 5′-half of COM (uCOM) forms four retarded complexes with HeLa or Hep-G2 nuclear proteins (UEF-1–4). We have identified the binding sequence for UEF-4 and generated uCOM elements uniquely mutated in the UEF-4-binding site or uniquely binding UEF-4. Introduction of these and other mutations in the context of the urokinase enhancer showed that all uCOM sites are important for enhancer activity but that UEF-4 and UEF-1 plus UEF-2/3 can substitute for each other, suggesting functional redundancy of urokinase enhancer factors. UEF-4 was purified from HeLa nuclear extract by affinity chromatography and shown to contain two polypeptides of 105 and 65 kDa, respectively, of which at least the former was endowed with DNA binding activity

SHORT-TERM RESPONSE OF SOIL MICROORGANISMS TO ESSENTIAL OILS WITH ALLELOPATHIC POTENTIAL EXTRACTED FROM MEDITERRANEAN PLANTS

Essential oils (EOs) with allelopathic compounds have been used to reduce or avoid weed germination and growth. The aim of this study was to evaluate the potential phytotoxic effects of EOs extracted from different Mediterranean plants on soil microbial biomass and activity. EOs were extracted from leaves of Eucalyptus camaldulensis Dehnh (EUC); Eriocephalus africanus L. (ERI); Thymus capitatus (L.) Hoffmanns. & Link (TCP); Citrus reticulata Blanco var. ‘Clemenules’ (TAN) and Citrus limon (L.) Osbeck var. ‘Eureka’ (LEM). Each EO was supplied to pots containing 560 g of soil at three different doses (low, medium, high). After 15, 30, 90, 120 days the supply of EOs, soils were destructively analyses for microbial biomass carbon (MBC) and microbial respiration. EOs extracted from E. camaldulensis (EUC), C. limon (LEM) and T. capitatus (TCP), at the highest concentration decreased MBC up to 30 days since their addition, with no further effects at two last samplings. EOs extracted from ERI and TAN did not affect MBC. Soil respiration was not affected by any experimental factor, whereas the metabolic quotient was increased by EO extracted from TCP. Our results suggested that essential oils with allelopathic potential extracted from mediterranean plants can negatively affect soil microorganisms and, consequently, their use as herbicides should take into account these findings

Cytosolic Sequestration of Prep1 Influences Early Stages of T Cell Development

Objective: Prep1 and Pbx2 are the main homeodomain DNA-binding proteins of the TALE (three amino acid loop extension) family expressed in the thymus. We previously reported reduced Pbx2 expression and defective thymocyte maturation in Prep1 hypomorphic mice. To further investigate the role of this homeodomain DNA-binding protein in T cell development, we generated transgenic mice expressing the N-terminal fragment of Pbx1 (Pbx1NT) under the control of the Lck proximal promoter. Principal Findings: Pbx1NT causes Prep1 cytosolic sequestration, abolishes Prep1-dependent DNA-binding activity and results in reduced Pbx2 expression in developing thymocytes. Transgenic thymi reveal increased numbers of CD4 2 CD8 2 CD44 2 (DN3 and DN4) thymocytes, due to a higher frequency of DN2 and DN4 Pbx1NT thymocytes in the S phase. Transgenic thymocytes however do not accumulate at later stages, as revealed by a normal representation of CD4/CD8 double positive and single positive thymocytes, due to a higher rate of apoptotic cell death of DN4 Pbx1NT thymocytes. Conclusion: The results obtained by genetic (Prep1 hypomorphic) and functional (Pbx1NT transgenic) inactivation of Prep

Analysis of expression of Pbx1NT transgene, Prep1 and Pbx2 proteins and Prep1-Pbx DNA-binding activity in Pbx1NT TG mice.

<p>(A) Western blot analysis of Pbx1NT expression in the thymus of <i>Pbx1NT TG mice</i>. Cytoplasmic extracts (30 µg) of total thymus of Pbx1NT TG mice (TG) and their wild-type controls (WT) were blotted onto PVDF membranes and incubated with anti-Pbx1 antibody. Anti-β-actin antibody was used as loading control. (B) RT-PCR analysis of Pbx1NT expression in sorted DN1, DN3 as well as in unsorted (TOTAL) thymocytes of <i>Pbx1NT TG mice</i>. Total RNA was isolated from the DN1, DN3 and total thymocytes and reverse transcribed. PCR amplification was performed using Pbx1NT-specific primers for 30 cycles and β-actin primers for 25 cycles. (C) Nuclear (Nuc) and cytoplasmic (Cyt) extracts of Pbx1NT TG mice (TG) and their wild-type controls (WT) were blotted onto PVDF membranes and incubated with anti-Prep1 and anti-Pbx2 antibodies. Anti-β-actin and anti-HMG1 antibodies were used as loading controls for cytoplasmic and nuclear fractions. (D) Total RNA was isolated from thymocytes of two Pbx1NT TG mice (TG) and their wild-type controls (WT) and reverse transcribed. PCR amplification was performed using Prep1 and Pbx2 -specific primers for 28 cycles and β-actin primers for 25 cycles. (E) Electrophoretic mobility shift assays were performed with the radiolabeled O1 double-stranded oligonucleotide, incubated with nuclear extracts from the thymi of wild-type (WT) or Pbx1NT TG (TG) mice without (−) or with antibodies against Prep1, Pbx1, Pbx2, Pbx3, Pbx4, Meis1 or Meis2. Pbx2-Prep1 arrow indicates the migration of complexes formed between Prep1 and Pbx2. SS, supershift.</p

Analysis of expression of Prep1, Pbx, and Meis proteins in thymus of FVB/N wt mice.

<p>Nuclear (Nuc) and cytoplasmic (Cyt) extracts of thymocytes (TH) of FVB/N wild-type mice were blotted onto nitrocellulose membranes and incubated with anti-Prep1 (A), anti-Pbx1b and anti-Pbx2 (B), anti-Pbx1 (Pbx1a and Pbx1b) and anti-Meis (C) antibodies. Anti-Sp1, anti-nucleolin, anti-β-actin and anti-α-vinculin antibodies were used as loading controls for nuclear and cytoplasmic extracts. A nuclear extracts of HeLa cell line or P19 cell line induced with retinoic acid (10⁻⁷ M) for 24 h were used as positive controls for the expression of Prep1, Pbx1 and Meis proteins. (D) RT-PCR analysis of Pbx1 (Pbx1a and Pbx1b), Pbx2 and Pbx3 (Pbx3a and Pbx3b) expression. Total RNA was isolated from thymocytes of FVB/N mice. Serial dilutions (1∶5) of cDNA were amplified using primers specific for Pbx1, Pbx2 and Pbx3. Primers for β-actin were used as a control.</p

T cell populations in wild-type (wt) and <i>Pbx1NT</i> (tg) mice

<p>Data are means (bold)±s.e.m. (in parenthesis)</p>*<p>million of cells per organ</p><p>N.D. non determined</p

Analysis of proliferation of thymocytes of Pbx1NT TG mice.

<p>Percentage of BrdU-positive DN1, DN2, DN3 and DN4 thymocytes after <i>in-vivo</i> BrdU staining (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002424#s2" target="_blank">Matherial and Methods</a>) is shown. The cells were from wild-type mice (open bars) or from Pbx1NT TG mice (black bars). (B) Cell cycle analysis of electronically gated DN2, DN3 and DN4 was performed with BrdU-flow kit (BD Biosciences, San Jose, CA) according to the manufacturer's recommendations. The cells were from wild-type mice (open bars) or from Pbx1ΝΤ TG mice (black bars).</p

Flow cytometric analysis of thymocytes from Pbx1NT TG mice (TG) and their wild-type controls (WT).

<p>(A) Thymocytes were stained with PE-conjugated anti-CD4, CyChrome-conjugated anti-CD8, and FITC-conjugated anti-TCRβ antibodies. Viable cells were gated according to their forward scatter (FCS) and side scatter (SCS) characteristics. One representative of 8 independent experiments is shown. (B) Absolute numbers of DN, DP and SP (CD4⁺ and CD8⁺) cells. The cells were from wild-type mice (open bars) or from Pbx1NT TG mice (black bars). (C) Flow cytometric analysis of DN (CD4⁻CD8⁻) thymocytes from Pbx1NT TG mice and their wild-type controls. DN cells were enriched by rabbit anti-CD4 and anti-CD8 complement-mediated lysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002424#s2" target="_blank">Materials and Methods</a>). DN thymocytes were stained with PE-conjugated anti-CD44, APC-conjugated anti-CD25 antibodies and CyChrome-conjugated anti-CD4 and anti-CD8 antibodies to negatively gate residual CD4 and CD8 -positive cells. Viable cells were gated according to their FCS and SCS characteristics. One representative of 8 independent experiments is shown. (D) Absolute numbers of DN1, DN2, DN3, and DN4 within DN cells. The cells were from wild-type mice (open bars) or from Pbx1ΝΤ TG mice (black bars). Values that were significantly different are indicated as follows: *, P<0.05; ***, P<0.001. (E) The same as in (C) for TN (CD4⁻CD8⁻CD3⁻) thymocytes. (F) The same as in (D) for TN thymocytes.</p

Analysis of apoptosis of thymocytes of Pbx1NT TG mice.

<p>Purified DN thymocytes from Pbx1ΝΤ TG mice and their wild-type controls were stained with PE-conjugated anti-CD44, APC-conjugated anti-CD25, FITC-conjugated annexin V, and 7AAD prior to FACS analysis. The percentage of annexinV-positive 7AAD-negative DN3 and DN4 thymocytes <i>ex vivo</i> (0h) and after 24 hours of incubation at 37°C (24 h) is shown. The cells were from wild-type mice (open bars) or from Pbx1ΝΤ TG mice (black bars). Values that were significantly different are indicated as follows: *, P<0.05.</p