Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.

Impact of Sn/F Pre-Treatments on the Durability of Protective Coatings against Dentine Erosion/Abrasion.

For preventing erosive wear in dentine, coating with adhesives has been suggested as an alternative to fluoridation. However, clinical studies have revealed limited efficacy. As there is first evidence that Sn(2+) increases bond strength of the adhesive Clearfil SE (Kuraray), the aim of the present study was to investigate whether pre-treatment with different Sn(2+)/F(-) solutions improves the durability of Clearfil SE coatings. Dentine samples (eight groups, n=16/group) were freed of smear layer (0.5% citric acid, 10 s), treated (15 s) either with no solution (control), aminefluoride (AmF, 500 ppm F(-), pH 4.5), SnCl2 (800/1600 ppm Sn(2+); pH 1.5), SnCl2/AmF (500 ppm F(-), 800 ppm Sn(2+), pH 1.5/3.0/4.5), or Elmex Erosion Protection Rinse (EP, 500 ppm F-, 800 ppm Sn(2+), pH 4.5; GABA International), then rinsed with water (15 s) and individually covered with Clearfil SE. Subsequently the specimens were subjected to an erosion/abrasion protocol consisting of 1320 cycles of immersion in 0.5% citric acid (5 °C/55 °C; 2 min) and automated brushing (15 s, 200 g, NaF-toothpaste, RDA 80). As the coatings proved stable up to 1320 cycles, 60 modified cycles (brushing time 30 min/cycle) were added. Wear was measured profilometrically. After SnCl2/AmF, pH 4.5 or EP pre-treatment all except one coating survived. In the other groups, almost all coatings were lost and there was no significant difference to the control group. Pre-treatment with a Sn(2+)/F(-) solution at pH 4.5 seems able to improve the durability of adhesive coatings, rendering these an attractive option in preventing erosive wear in dentine.

Experimental models for p53, hepatitis B and aflatoxin hepatocarcinogenesis

The major risk factors for liver cancer in Southeast Asia: HBV infection, aflatoxin exposure and p53 expression/mutation, were examined in experimental models. Four groups were examined for development of hepatocellular carcinoma (HCC) with and without neonatal exposure to aflatoxin (AFB$\sb1)$: (Group I.) Transgenic HBsAg mice with one p53 allele. (Group II) Transgenic HBsAg mice with two p53 alleles. (Group III) Non-transgenic litter mates with one p53 allele. (Group IV) Non-transgenic litter mates with two p53 alleles. HCC developed in Group I animals exposed to aflatoxin at an earlier time and were of a higher grade than those seen later in other groups. These results provide an explanation for as to why p53 is a target for deletion and/or mutation in human HCC especially when found in high risk areas where HBV infection and Aflatoxin B1 food contamination is high, and nicely illustrates a synergistic interaction among these three factors. None of the tumors analyzed had loss or mutation in the p53 gene.^ To determine the significance of the specific p53ser249 mutation found in HBV/aflatoxin associated human hepatomas in an in-vivo experimental model using transgenic mice, a two-nucleotide change in the mouse p53 gene at amino acid position 246, which is equivalent to that of 249 in human p53, was introduced. Transgenic mice with mutant p53 controlled by the albumin promoter were generated and shown to express the p53ser246 mutant RNA and protein specifically in liver. Three groups were examined for development of HCC with and without neonatal exposure to aflatoxin: (Group V) Transgenic p53ser246 mice with two p53 alleles. (Group VI) Transgenic p53ser246 mice with one p53 allele. (Group VII) Double transgenic for p53ser246 and HBsAg with two p53 alleles. One hundred percent of male mice with the three risk factors injected with aflatoxin developed high grade liver tumors, compared to 66.6% from group VI and only 14.2% of group V suggesting synergistic interaction between HBsAg and this particular ser246 p53 mutation.^ In order to examine the growth properties of hepatocytes and correlation with p53 loss and/or mutation, cell proliferation and ploidy analysis of liver from normal heterozyous, homozygous null mice and from transgenic mutant p53ser246, mice were studied. Loss of wild-type p53 increased G1/G0 ratios of cells as well as proliferation and decreased cell ploidy. The mutant p53ser246 did not show a significant effect on cell ploidy or proliferation. However a striking 5-10X increase in G1/G0 ratio suggests that this specific mutation specifically induces G0 to G1 transition, which in turn further predisposes hepatocytes to the damaging effect of Aflatoxin. (Abstract shortened by UMI.) ^

A reflection suppression technique for far field antenna measurements

This paper presents a reflection suppression technique for far field antenna measurements. The technique is based on a source reconstruction over a surface greater than the antenna itself. To be able to perform the reflection construction the next steps are required: the complete far field antenna pattern is obtained through interpolation of the acquired cuts, the currents are obtained through a holographic technique, the field out of the antenna area is filtered, and the pattern is reconstructed. The algorithm is used with measurements in the LEHA-UPM antenna measurement facilities and in the outdoor far field facility of LIT INPE in Brazil.

Analysis and suppression of reflections in far-field antenna measurement ranges

This paper presents the analysis of the reflections in two kind of spherical far field ranges: one if the classical acquisition where the AUT is rotated and the second one corresponds to the systems where the AUT is fixed and the antenna probe is rotated. In large far field systems this is not possible, but this can be used to the measurement of small antennas, for instance, with the SATIMO StarGate system. In both cases, it is assumed that only one frequency is acquired and the results should be improved cut by cut, in order not to lose the advantages or far field measurements. Finally, some practical results are studied using measurements of one antenna in the outdoor far field facility of LIT INPE in Brazil.

Be Careful Where You Smile : Culture Shapes Judgments of Intelligence and Honesty of Smiling Individuals

We thank all the supporting team-members involved in the translation procedures and data collections. Research was supported by the Polish NCN Grant 2011/03/N/HS6/05112 (K.K.) and Chinese NNSF Grant 31200788 (C.X).

Down-Regulation of Platelet Endothelial Cell Adhesion Molecule-1 Results in Thrombospondin-1 Expression and Concerted Regulation of Endothelial Cell Phenotype

bEND.3 cells are polyoma middle T-transformed mouse brain endothelial cells that express very little or no thrombospondin-1, a natural inhibitor of angiogenesis, but express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) that localizes to sites of cell–cell contact. Here, we have examined the role of PECAM-1 in regulation of bEND.3 cell proliferation, migration, morphogenesis, and hemangioma formation. We show that down-regulating PECAM-1 expression by antisense transfection of bEND.3 cells has a dramatic effect on their morphology, proliferation, and morphogenesis on Matrigel. There is an optimal level for PECAM-1 expression such that high levels of PECAM-1 inhibit, whereas moderate levels of PECAM-1 stimulate, endothelial cell morphogenesis. The down-regulation of PECAM-1 in bEND.3 cells resulted in reexpression of endogenous thrombospondin-1 and its antiangiogenic receptor CD36. The expression of the vascular endothelial growth factor receptors flk-1 and flt-1, as well as integrins and metalloproteinases (which are involved in angiogenesis), were also affected. These observations are consistent with the changes observed in proliferation, migration, and adhesion characteristics of the antisense-transfected bEND.3 cells as well as with their lack of ability to form hemangiomas in mice. Thus, a reciprocal relationship exists between thrombospondin-1 and PECAM-1 expression, such that these two molecules appear to be constituents of a “switch” that regulates in concert many components of the angiogenic and differentiated phenotypes of endothelial cells.

A Role for Cadherin-5 in Regulation of Vascular Endothelial Growth Factor Receptor 2 Activity in Endothelial Cells

FLK-1/vascular endothelial growth factor receptor 2 (VEGFR-2) is one of the receptors for VEGF. In this study we examined the effect of cell density on activation of VEGFR-2. VEGF induces only very slight tyrosine phosphorylation of VEGFR-2 in confluent (95–100% confluent) pig aortic endothelial (PAE) cells. In contrast, robust VEGF-dependent tyrosine phosphorylation of VEGFR-2 was observed in cells plated in sparse culture conditions (60–65% confluent). A similar cell density-dependent phenomenon was observed in different endothelial cells but not in NIH-3T3 fibroblast cells expressing VEGFR-2. Stimulating cells with high concentrations of VEGF or replacing the extracellular domain of VEGFR-2 with that of the colony-stimulating factor 1 receptor did not alleviate the sensitivity of VEGFR-2 to cell density, indicating that the confluent cells were probably not secreting an antagonist to VEGF. Furthermore, in PAE cells, ectopically introduced platelet-derived growth factor α receptor could be activated at both high and low cell density conditions, indicating that the density effect was not universal for all receptor tyrosine kinases expressed in endothelial cells. In addition to lowering the density of cells, removing divalent cations from the medium of confluent cells potentiated VEGFR-2 phosphorylation in response to VEGF. These findings suggested that cell–cell contact may be playing a role in regulating the activation of VEGFR-2. To this end, pretreatment of confluent PAE cells with a neutralizing anti-cadherin-5 antibody potentiated the response of VEGFR-2 to VEGF. Our data demonstrate that endothelial cell density plays a critical role in regulating VEGFR-2 activity, and that the underlying mechanism appears to involve cadherin-5.