Effect of Phosphorylation on EGFR Dimer Stability Probed by Single-Molecule Dynamics and FRET/FLIM

Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (k_off). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the k_off of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell’s proliferation potential.     

Optimizing culture conditions of a porcine epithelial cell line IPEC-J2 through a histological and physiological characterization

The high similarity between pigs and humans makes pigs a good gastrointestinal (GI) model for humans. Recently an epithelial cell line originating from the jejunum of pig (IPEC-J2) became available. Once validated, this model can be used to investigate the complex interactions occurring in the intestine. The advantages of using IPEC-J2 as in vitro model of the GI tract are the high resemblance between humans and pigs, and the ease of extrapolating in vitro to in vivo characteristics. In this study, the IPEC-J2 cells were functionally characterized by measuring the trans-epithelial electrical resistance (TEER), and by histological and ultrastructural studies. IPEC-J2 cells grown on six different permeable support systems, were investigated. The Transwell^®-COL collagen-coated membrane (1.12 cm²) showed the best results concerning time efficiency and TEER values. The optimum seeding density of 12 × 10⁵ cells/mL ensured that after 9 days of differentiation a confluent monolayer was formed. The decrease in TEER values after a maximum had been reached, coincided with the ultrastructural development of apical microvilli. We conclude that IPEC-J2 cells grown on collagen-coated membranes represent a valuable in vitro model system for the small intestinal epithelium which can be of great interest for intestinal research.     

Mitotic exit control: a space and time odyssey

The mitotic exit network (MEN), a protein kinase cascade under the switch-like control of the small GTPase Tem1, triggers exit from mitosis in budding yeast. Now it emerges that signals from both Tem1 and the yeast Polo kinase Cdc5 converge onto the MEN kinase Cdc15 to accurately restrict MEN activation to late mitosis.     

New concepts of IL-10-induced lung fibrosis: fibrocyte recruitment and M2 activation in a CCL2/CCR2 axis

IL-10 is most commonly recognized as an anti-inflammatory cytokine possessing immunosuppressive effects necessary for regulated resolution of proinflammation. However, its role in the development of fibrosis during inflammatory resolution has not been clear. Few prior studies have linked IL-10 with the inhibition of fibrosis principally on the basis of regulating inflammation thought to be driving fibroproliferation. In contrast, in a model of long-term overexpression of IL-10, we observed marked induction of lung fibrosis in mice. The total cell number retrieved by bronchoalveolar lavage (BAL) increased 10-fold in the IL-10 overexpression (IL-10 OE) mice, with significant infiltration of T and B lymphocytes and collagen-producing cells. The presence of increased fibrocytes, isolated from collagenase-digested lungs, was identified by flow cytometry using dual staining of CD45 and collagen 1. Quantitative PCR analysis on an array of chemokine/chemokine receptor genes showed that receptor CCR2 and its ligand, CCL2, were highly upregulated in IL-10 OE mice, suggesting that IL-10-induced fibrocyte recruitment was CCL2/CCR2 specific. Given the prior association of alternatively activated (M(2)) macrophages with development of fibrosis in other disease states, we also examined the effect of IL-10 OE on the M(2) macrophage axis. We observed significantly increased numbers of M(2) macrophages in both BAL and whole lung tissue from the IL-10 OE mice. Administration of rabbit anti-CCL2 antiserum to IL-10 OE mice for three consecutive weeks significantly decreased fibrosis as evidenced by lung hydroxyproline content, compared with mice that received preimmune rabbit serum. These results indicate that overexpression of IL-10 induces fibrosis, in part, by fibrocyte recruitment and M(2) macrophage activation, and likely in a CCL2/CCR2 axis.     

Delivery of prolamins to the protein storage vacuole in maize aleurone cells

Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.     

Resveratrol inhibits proliferation and survival of Epstein Barr virus-infected Burkitt's lymphoma cells depending on viral latency program

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenolic natural product, shows chemopreventive properties against several cancers, heart diseases, inflammation, and viral infections. Epstein Barr virus (EBV), a γ-herpesvirus, contributes to the development of several human cancers including Burkitt's lymphoma (BL). In this study, we asked whether treatment with resveratrol would affect the viability of EBV-positive BL cells displaying different forms of latency. We report here that resveratrol, regardless of EBV status, induces caspase-dependent apoptosis by arresting cell-cycle progression in G(1) phase. However, resveratrol strongly induced apoptosis in EBV(-) and latency I EBV(+) cells, whereas latency II and latency III EBV(+) BL cells showed a survival advantage that increased with the extent of the pattern of viral gene expression. Resveratrol-induced cell-cycle arrest and apoptosis occurred in association with induction of p38 MAPK phosphorylation and suppression of ERK1/2 signaling pathway. Moreover, NF-κB DNA-binding activity was inhibited in all BL lines except EBV(+) latency III cells. LMP1 oncogene, which is expressed in latency III phenotype, is involved with the higher resistance to the antiproliferative effect of resveratrol because siRNA-mediated inhibition of LMP1 greatly increased the sensitivity of latency III BL cells as well as that of lymphoblastoid cell lines to the polyphenol. We propose that a combined resveratrol/siRNA strategy may be a novel approach for the treatment of EBV-associated B-cell malignancies in which the viral pattern of gene expression has been defined.     

Assessing ecological risk through automated drainage extraction and watershed delineation

Decision makers are frequently involved in projects requiring ecological risk definition, which are inherent to biological conservation process. It is important to recognize these risks in order to invest wisely in the management and protection of biological resources. In this matter, Geographic Information System tools and remote sensing data have been used frequently as important components in planning and management of conservation units, Rabus et al. (2003), Valeriano et al. (2009) and Valeriano et al. (2010) stressed the advantages of using data that were gathered during the Shuttle Radar Topographic Mission (SRTM) for biological and geomorphologic purposes. For Brazil's national territory, the SRTM data were refined (Valeriano, 2008) and offered as free access on the TOPODATA Project website (http://www.dsr.inpe.br/topodata) where geomorphometric information (including elevation data) at a resolution of 30 m are provided. The aim of this paper is to demonstrate an example of how TOPODATA products have been applied in order to determine the ecological risk of the border of a Conservation Unit, located in the State of São Paulo—Brazil, in the Brazilian Atlantic Forest, using automated drainage network and watershed extraction. A comparison between SRTM, TOPODATA, and ASTER DEM was carried out, showing an advantage of TOPODATA drainage network product. The vectors generated using this data are more similar to the official drainage network vectors than the drainage network extracted using ASTER-DEM or SRTM. The network product generated using ASTER-DEM produced many commission errors and the one generated using SRTM produced a poor network, with generalized vectors, less detailed than the others. The results showed that using the TOPODATA Project‘s Digital Elevation Model (DEM) can provide important data for ecological analysis and significant additional information for decision making, regarding drainage networks and watershed features. The produced map for border ecological risk showed to fit perfectly to the field work analyses, produced in other works. Furthermore, the extracted watershed polygons might furnish important information unrevealing best conservation unit boundaries, which means more efficient management and best biological conservation results.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after the incubation of 2'-deoxyguanosine (dGuo) with ChOOH and Cu(2+). In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.