The Y-box factor ZONAB/DbpA associates with GEF-H1/Lfc and mediates Rho-stimulated transcription

Epithelial tight junctions recruit different types of signalling proteins that regulate cell proliferation and differentiation. Little is known about how such proteins interact functionally and biochemically with each other. Here, we focus on the Y-box transcription factor ZONAB (zonula occludens 1-associated nucleic-acid-binding protein)/DbpA (DNA-binding protein A) and the Rho GTPase activator guanine nucleotide exchange factor (GEF)-H1/Lbc's first cousin, which are two tight-junction-associated signalling proteins that regulate proliferation. Our data show that the two proteins interact and that ZONAB activity is Rho-dependent. Overexpression of GEF-H1 induces accumulation of ZONAB in the nucleus and activates transcription. Microtubule-affinity regulating kinase/partition-defective-1, another type of GEF-H1-associated signalling protein, remains in the cytoplasm and partially co-localizes with the exchange factor. GEF-H1 and ZONAB are required for expression of endogenous cyclin D1, a crucial RhoA signalling target gene, and GEF-H1-stimulated cyclin D1 promoter activity requires ZONAB. Our data thus indicate that GEF-H1 and ZONAB form a signalling module that mediates Rho-regulated cyclin D1 promoter activation and expression

Holey barrier: claudins and the regulation of brain endothelial permeability

Endothelial tight junctions (TJs)* are an important functional part of the blood-brain barrier (BBB). In this issue, Nitta et al. (2003) demonstrate that claudin-5, a transmembrane protein of TJs, is a critical determinant of BBB permeability in mice. Unexpectedly, knockout of claudin-5 did not result in a general breakdown of TJs but in a selective increase in paracellular permeability of small molecules. This suggests that the BBB can be manipulated to allow selective diffusion of small molecules and makes claudin-5 a possible target for the development of drugs for this purpose

Application of Risk Management for Control and Monitoring Systems

This paper presents an application of the state of the art and new trends for risk management of safety-related control and monitoring systems, currently applied in the industry. These techniques not only enable to manage safety and reliability issues but they also help in the control of quality and economic factors affected by the availability and maintenance of the system. The method includes an unambiguous definition of the system in terms of functions and a systematic analysis of hazardous situations, undesired events and possible malfunctions. It also includes the identification and quantification of the risk associated to the system. The required risk reduction is specified in terms of safety integrity levels. The safety integrity level results in requirements, preventive measures, possible improvements and recommendations to assure the satisfactory management of the risk

The ZO-1–associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density

Epithelial tight junctions regulate paracellular permeability, restrict apical/basolateral intramembrane diffusion of lipids, and have been proposed to participate in the control of epithelial cell proliferation and differentiation. Previously, we have identified ZO-1–associated nucleic acid binding proteins (ZONAB), a Y-box transcription factor whose nuclear localization and transcriptional activity is regulated by the tight junction–associated candidate tumor suppressor ZO-1. Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells. Transfection of wild-type or ZONAB-binding fragments of ZO-1 reduced proliferation as well as nuclear ZONAB pools, indicating that promotion of proliferation by ZONAB requires its nuclear accumulation. Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density. ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4. Thus, our data indicate that tight junctions can regulate epithelial cell proliferation and cell density via a ZONAB/ZO-1–based pathway. Although this regulatory process may also involve regulation of transcription by ZONAB, our data suggest that one mechanism by which ZONAB and ZO-1 influence proliferation is by regulating the nuclear accumulation of CDK4

Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2

Background: Tight junctions are required for epithelial barrier formation and participate in the regulation of signalling mechanisms that control proliferation and differentiation. ZO-1 is a tight junction-associated adaptor protein that regulates gene expression, junction assembly and epithelial morphogenesis. We have previously demonstrated that the heat shock protein Apg-2 binds ZO-1 and thereby regulates its role in cell proliferation. Here, we addressed the question whether Apg-2 is also important for junction formation and epithelial morphogenesis.Results: We demonstrate that depletion of Apg-2 by RNAi in MDCK cells did not prevent formation of functional tight junctions. Similar to ZO-1, however, reduced expression of Apg-2 retarded de novo junction assembly if analysed in a Ca-switch model. Formation of functional junctions, as monitored by measuring transepithelial electrical resistance, and recruitment of tight and adherens junction markers were retarded. If cultured in three dimensional extracellular matrix gels, Apg-2 depleted cells, as previously shown for ZO-1 depleted cells, did not form hollow polarised cysts but poorly organised, irregular structures.Conclusion: Our data indicate that Apg-2 regulates junction assembly and is required for normal epithelial morphogenesis in a three-dimensional culture system, suggesting that Apg-2 is an important regulator of epithelial differentiation. As the observed phenotypes are similar to those previously described for ZO-1 depleted cells and depletion of Apg-2 retards junctional recruitment of ZO-1, regulation of ZO-1 is likely to be an important functional role for Apg-2 during epithelial differentiation

Small and large intestine express a truncated Dab1 isoform that assembles in cell-cell junctions and co-localizes with proteins involved in endocytosis

Disabled-1 (Dab1) is an essential intracellular adaptor protein in the reelin pathway. Our previous studies in mice intestine showed that Dab1 transmits the reelin signal to cytosolic signalling pathways. Here, we determine the Dab1 isoform expressed in rodent small and large intestine, its subcellular location and co-localization with clathrin, caveolin-1 and N-Wasp. PCR and sequencing analysis reveal that rodent small and large intestine express a Dab1 isoform that misses three (Y198, Y200 and Y220) of the five tyrosine phosphorylation sites present in brain Dab1 isoform (canonical) and contains nuclear localization and export signals. Western blot assays show that both, crypts, which shelter progenitor cells, and enterocytes express the same Dab1 isoform, suggesting that epithelial cell differentiation does not regulate intestinal generation of alternatively spliced Dab1 variants. They also reveal that the canonical and the intestinal Dab1 isoforms differ in their total degree of phosphorylation. Immunostaining assays show that in enterocytes Dab1 localizes at the apical and lateral membranes, apical vesicles, close to adherens junctions and desmosomes, as well as in the nucleus; co-localizes with clathrin and with N-Wasp but not with caveolin-1, and in Caco-2 cells Dab1 localizes at cell-to-cell junctions by a Ca2+-dependent process. In conclusion, the results indicate that in rodent intestine a truncated Dab1 variant transmits the reelin signal and may play a role in clathrin-mediated apical endocytosis and in the control of cell-to-cell junction assembly. A function of intestinal Dab1 variant as a nucleocytoplasmic shuttling protein is also inferred from its sequence and nuclear location.Junta de Andalucía CTS 5884Ministerio de Educación y Ciencia AP2007-04201European Molecular Biology Organization ASTF45-201

Upconversion cooling of Er-doped low-phonon fluorescent solids

We report on a novel mechanism for laser cooling of fluorescent solids based on infrared-to-visible upconversion often found in rare-earth-doped low-phonon materials. This type of optical cooling presents some advantages with regards to conventional anti-Stokes cooling. Among them, it allows to obtain cooling in a broader range of frequencies around the barycenter of the infrared emitting band.Comment: 4 pages, 1 figur

Spectroscopy of thulium and holmium heavily doped tellurite glasses

In this study, we report spectroscopic properties of Tm3þ and Ho3þ codoped tellurite glasses over a wide dopant concentration range in order to assess their potential laser performance under 790 nm diode laser excitation. The impact of Tm3þ and Ho3þ concentrations is investigated to identify specific candidates for fiber laser operation. The emission cross section is calculated and discussed, as well as the gain coefficient of this type of glasses. Energy transfer microparameters and critical ion distances are determined for 3H4, 3F4 (Tm3þ), and 5I7 (Ho3þ) emission levels in the framework of diffusionlimited regime and dipole-dipole interaction. We also report thermal properties of tested glasse