Nuclear translocation and DNA recognition signals colocalized within the bZIP domain of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB.

CREB is a cAMP-responsive nuclear DNA-binding protein that binds to cAMP response elements and stimulates gene transcription upon activation of the cAMP signalling pathway. The protein consists of an amino-terminal transcriptional transactivation domain and a carboxyl-terminal DNA-binding domain (bZIP domain) comprised of a basic region and a leucine zipper involved in DNA recognition and dimerization, respectively. Recently, we discovered a testis-specific transcript of CREB that contains an alternatively spliced exon encoding multiple stop codons. CREB encoded by this transcript is a truncated protein lacking the bZIP domain. We postulated that the antigen detected by CREB antiserum in the cytoplasm of germinal cells is the truncated CREB that must also lack its nuclear translocation signal (NTS). To test this hypothesis we prepared multiple expression plasmids encoding carboxyl-terminal deletions of CREB and transiently expressed them in COS-1 cells. By Western immunoblot analysis as well as immunocytochemistry of transfected cells, we show that CREB proteins truncated to amino acid 286 or shorter are sequestered in the cytoplasm, whereas a CREB of 295 amino acids is translocated into the nucleus. Chimeric CREBs containing a heterologous NTS fused to the first 248 or 261 amino acids of CREB are able to drive the translocation of the protein into the nucleus. Thus, the nine amino acids in the basic region involved in DNA recognition between positions 287 and 295 (RRKKKEYVK) of CREB contain the NTS. Further, mutation of the lysine at position 290 in CREB to an asparagine diminishes nuclear translocation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in the TGFβ binding-protein-like domain 5 of FBN1 are responsible for acromicric and geleophysic dysplasias.

Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although AD has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. After exome sequencing in GD and AD cases, we selected fibrillin 1 (FBN1) as a candidate gene, even though mutations in this gene have been described in Marfan syndrome, which is characterized by tall stature and arachnodactyly. We identified 16 heterozygous FBN1 mutations that are all located in exons 41 and 42 and encode TGFβ-binding protein-like domain 5 (TB5) of FBN1 in 29 GD and AD cases. Microfibrillar network disorganization and enhanced TGFβ signaling were consistent features in GD and AD fibroblasts. Importantly, a direct interaction between ADAMTSL2 and FBN1 was demonstrated, suggesting a disruption of this interaction as the underlying mechanism of GD and AD phenotypes. Although enhanced TGFβ signaling caused by FBN1 mutations can trigger either Marfan syndrome or GD and AD, our findings support the fact that TB5 mutations in FBN1 are responsible for short stature phenotypes.

Mycobacterium tuberculosis subverts innate immunity to evade specific effectors.

The macrophage is the niche of the intracellular pathogen Mycobacterium tuberculosis. Induction of macrophage apoptosis by CD4(+) or CD8(+) T cells is accompanied by reduced bacterial counts, potentially defining a host defense mechanism. We have already established that M. tuberculosis-infected primary human macrophages have a reduced susceptibility to Fas ligand (FasL)-induced apoptosis. To study the mechanisms by which M. tuberculosis prevents apoptotic signaling, we have generated a cell culture system based on PMA- and IFN-gamma-differentiated THP-1 cells recapitulating the properties of primary macrophages. In these cells, nucleotide-binding oligomerization domain 2 or TLR2 agonists and mycobacterial infection protected macrophages from apoptosis and resulted in NF-kappaB nuclear translocation associated with up-regulation of the antiapoptotic cellular FLIP. Transduction of a receptor-interacting protein-2 dominant-negative construct showed that nucleotide-binding oligomerization domain 2 is not involved in protection in the mycobacterial infection system. In contrast, both a dominant-negative construct of the MyD88 adaptor and an NF-kappaB inhibitor abrogated the protection against FasL-mediated apoptosis, showing the implication of TLR2-mediated activation of NF-kappaB in apoptosis protection in infected macrophages. The apoptosis resistance of infected macrophages might be considered as an immune escape mechanism, whereby M. tuberculosis subverts innate immunity signaling to protect its host cell against FasL(+)-specific cytotoxic lymphocytes.

Disease-associated mutations in the actin-binding domain of filamin B cause cytoplasmic focal accumulations correlating with disease severity.

Dominant missense mutations in FLNB, encoding the actin-cross linking protein filamin B (FLNB), cause a broad range of skeletal dysplasias with varying severity by an unknown mechanism. Here these FLNB mutations are shown to cluster in exons encoding the actin-binding domain (ABD) and filamin repeats surrounding the flexible hinge 1 region of the FLNB rod domain. Despite being positioned in domains that bind actin, it is unknown if these mutations perturb cytoskeletal structure. Expression of several full-length FLNB constructs containing ABD mutations resulted in the appearance of actin-containing cytoplasmic focal accumulations of the substituted protein to a degree that was correlated with the severity of the associated phenotypes. In contrast, study of mutations leading to substitutions in the FLNB rod domain that result in the same phenotypes as ABD mutations demonstrated that with only one exception disease-associated substitutions, surrounding hinge 1 demonstrated no tendency to form actin-filamin foci. The exception, a substitution in filamin repeat 6, lies within a region previously implicated in filamin-actin binding. These data are consistent with mutations in the ABD conferring enhanced actin-binding activity but suggest that substitutions affecting repeats near the flexible hinge region of FLNB precipitate the same phenotypes through a different mechanism.

High capacity audio watermarking using the high frequency band of the wavelet domain

This paper proposes a novel high capacity robust audio watermarking algorithm by using the high frequency band of the wavelet decomposition at which the human auditory system (HAS) is not very sensitive to alteration. The main idea is to divide the high frequency band into frames and, for embedding, to change the wavelet samples depending on the average of relevant frame¿s samples. The experimental results show that the method has a very high capacity (about 11,000 bps), without significant perceptual distortion (ODG in [¿1 ,0] and SNR about 30dB), and provides robustness against common audio signal processing such as additive noise, filtering, echo and MPEG compression (MP3).

Efficient self-synchronised blind audio watermarking system based on time domain and FFT amplitude modification

Many audio watermarking schemes divide the audio signal into several blocks such that part of the watermark is embedded into each of them. One of the key issues in these block-oriented watermarking schemes is to preserve the synchronisation, i.e. to recover the exact position of each block in the mark recovery process. In this paper, a novel time domain synchronisation technique is presented together with a new blind watermarking scheme which works in the Discrete Fourier Transform (DFT or FFT) domain. The combined scheme provides excellent imperceptibility results whilst achieving robustness against typical attacks. Furthermore, the execution of the scheme is fast enough to be used in real-time applications. The excellent transparency of the embedding algorithm makes it particularly useful for professional applications, such as the embedding of monitoring information in broadcast signals. The scheme is also compared with some recent results of the literature.

Repurposing Social Tagging Data for Extraction of Domain-level Concepts

The World Wide Web, the world¿s largest resource for information, has evolved from organizing information using controlled, top-down taxonomies to a bottom up approach that emphasizes assigning meaning to data via mechanisms such as the Social Web (Web 2.0). Tagging adds meta-data, (weak semantics) to the content available on the web. This research investigates the potential for repurposing this layer of meta-data. We propose a multi-phase approach that exploits user-defined tags to identify and extract domain-level concepts. We operationalize this approach and assess its feasibility by application to a publicly available tag repository. The paper describes insights gained from implementing and applying the heuristics contained in the approach, as well as challenges and implications of repurposing tags for extraction of domain-level concepts.

The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways.

BACKGROUND: Activation of Fas (CD95) by its ligand (FasL) rapidly induces cell death through recruitment and activation of caspase-8 via the adaptor protein Fas-associated death domain protein (FADD). However, Fas signals do not always result in apoptosis but can also trigger a pathway that leads to proliferation. We investigated the level at which the two conflicting Fas signals diverge and the protein(s) that are implicated in switching the response. RESULTS: Under conditions in which proliferation of CD3-activated human T lymphocytes is increased by recombinant FasL, there was activation of the transcription factors NF-kappaB and AP-1 and recruitment of the caspase-8 inhibitor and FADD-interacting protein FLIP (FLICE-like inhibitory protein). Fas-recruited FLIP interacts with TNF-receptor associated factors 1 and 2, as well as with the kinases RIP and Raf-1, resulting in the activation of the NF-kappaB and extracellular signal regulated kinase (Erk) signaling pathways. In T cells these two signal pathways are critical for interleukin-2 production. Increased expression of FLIP in T cells resulted in increased production of interleukin-2. CONCLUSIONS: We provide evidence that FLIP is not simply an inhibitor of death-receptor-induced apoptosis but that it also mediates the activation of NF-kappaB and Erk by virtue of its capacity to recruit adaptor proteins involved in these signaling pathways.

Intracellular thiol-mediated modulation of epithelial sodium channel activity.

The epithelial sodium channel ENaC is physiologically important in the kidney for the regulation of the extracellular fluid volume, and in the lungs for the maintenance of the appropriate airway surface liquid volume that lines the pulmonary epithelium. Besides the regulation of ENaC by hormones, intracellular factors such as Na(+) ions, pH, or Ca(2+) are responsible for fast adaptive responses of ENaC activity to changes in the intracellular milieu. In this study, we show that ENaC is rapidly and reversibly inhibited by internal sulfhydryl-reactive molecules such as methanethiosulfonate derivatives of different sizes, the metal cations Cd(2+) and Zn(2+), or copper(II) phenanthroline, a mild oxidizing agent that promotes the formation of disulfide bonds. At the single channel level, these agents applied intracellularly induce the appearance of long channel closures, suggesting an effect on ENaC gating. The intracellular reducing agent dithiothreitol fully reverses the rundown of ENaC activity in inside-out patches. Our observations suggest that changes in intracellular redox potential modulate ENaC activity and may regulate ENaC-mediated Na(+) transport in epithelia. Finally, substitution experiments reveal that multiple cysteine residues in the amino and carboxyl termini of ENaC subunits are responsible for this thiol-mediated inhibition of ENaC.

Nanoparticles activate the NLR pyrin domain containing 3 (Nlrp3) inflammasome and cause pulmonary inflammation through release of IL-1α and IL-1β.

Nanoparticles are increasingly used in various fields, including biomedicine and electronics. One application utilizes the opacifying effect of nano-TiO(2), which is frequently used as pigment in cosmetics. Although TiO(2) is believed to be biologically inert, an emerging literature reports increased incidence of respiratory diseases in people exposed to TiO(2). Here, we show that nano-TiO(2) and nano-SiO(2), but not nano-ZnO, activate the NLR pyrin domain containing 3 (Nlrp3) inflammasome, leading to IL-1β release and in addition, induce the regulated release of IL-1α. Unlike other particulate Nlrp3 agonists, nano-TiO(2)-dependent-Nlrp3 activity does not require cytoskeleton-dependent phagocytosis and induces IL-1α/β secretion in nonphagocytic keratinocytes. Inhalation of nano-TiO(2) provokes lung inflammation which is strongly suppressed in IL-1R- and IL-1α-deficient mice. Thus, the inflammation caused by nano-TiO(2) in vivo is largely caused by the biological effect of IL-1α. The current use of nano-TiO(2) may present a health hazard due to its capacity to induce IL-1R signaling, a situation reminiscent of inflammation provoked by asbestos exposure.

Intracellular targeting of GLUT4 in transfected insulinoma cells: evidence for association with constitutively recycling vesicles distinct from synaptophysin and insulin vesicles.

In adipocytes and muscle cells, the GLUT4 glucose transporter isoform is present in intracellular vesicles which continuously recycle between an intracytoplasmic location and the plasma membrane. It is not clear whether the GLUT4-vesicles represent a specific kind of vesicle or resemble typical secretory granules or synaptic-like microvesicles. To approach this question, we expressed GLUT4 in the beta cell line RINm5F and determined its intracellular localization by subcellular fractionation and by immunofluorescence and immunoelectron microscopy. GLUT4 was not found in insulin granules but was associated with a subpopulation of smooth-surface vesicles present in the trans-Golgi region and in vesicular structures adjacent to the plasma membrane. In the trans-Golgi region, GLUT4 did not colocalize with synaptophysin or TGN38. Incubation of the cells with horseradish peroxidase (HRP) led to colocalization of HRP and GLUT4 in some endosomal structures adjacent to the plasma membrane and in occasional trans-Golgi region vesicles. When cells were incubated in the presence of Bafilomycin A, analysis by confocal microscopy revealed GLUT4 in numerous large spots present throughout the cytoplasm, many of which costained for TGN38 and synaptophysin. By immunoelectron microscopy, numerous endosomes were observed which stained strongly for GLUT4. Together our data demonstrate that ectopic expression of GLUT4 in insulinoma cells reveals the presence of a subset of vesicular structures distinct from synaptic-like vesicles and insulin secretory granules. Furthermore, they indicate that GLUT4 constitutively recycles between the plasma membrane and its intracellular location by an endocytic route also taken by TGN38 and synaptophysin.

Palaeotectonic and palaeogeographic evolution of the western Tethys and PeriTethyan domain (IGCP Project 369)

Project No. 369 (''Comparative Evolution of PeriTethyan Rift Basins'') of the International Geological Correlation Program produced a new palaeotectonic-palaeo-geographic atlas of the western PeriTethyan domain. The atlas contains more than two hundred new maps and documents grouped in nine regional sets (Iberia, Polish Trough, Eastern European and Scythian Platforms, Moesian Platform, Levant, Arabian Platform,, Northern Africa, NE Africa-NW Arabia, Libya-Pelagian Shelf plus a set of reconstructions for the whole western Tethys. The area, considered in the atlas stretches. from west to east, from the eastern Atlantic shores to the Urals and, from north to south, from the Baltic shield to equatorial Africa; the time span covered extends from the Late. Carboniferous to the Present. The dataset, resulting from an extensive cooperation between industrial and academic sources, is accessible interactively on a CD-ROM (Stampfli et al., 2001a) and includes legend, timetable, short explanatory notes, full references and additional supporting data. This dataset provides information on the development of the Tethyan realm in space and time. In particular, the relation between the Variscan and Cimmerian cycles in the Mediterranean realm is illustrated by numerous palaeogeographic and palaeotectonic maps.

Comparison of central macular thickness (CMT) measurement between time domain and spectral domain (SD) optical coherence tomography (OCT) and reproducibility of SD OCT in active neovascular AMD

Purpose: To evaluate the reproducibility of Cirrus-SD OCT measurements and to compare central macular thickness (CMT) measurements between TD-Stratus and SD-Cirrus OCT in patients with active exudative AMD. Methods: Consecutive case series of patients with active exudative AMD seen in the Medical Retina Department. Patients underwent 1 scan with Stratus (macular thickness map protocol) and 5 scans with Cirrus (Macular Cube protocol) at the same visit by the same experienced examiner. To be included, patients best-corrected visual acuity (BCVA) had to be >20/200 while all scans had to be of sufficient quality, well-centered and at least one Cirrus scan with CMT >300 microns. The repeatability of the SD Cirrus was estimated by using all 5 CMT measurements and the mean of the Cirrus measurements was compared with the CMT obtained by TD Stratus. Results: Cirrus OCT demonstrated high intraobserver repeatability at the central foveal region (ICC 96%). The mean of the CMT measurements was 321microns for Stratus and 387 microns for Cirrus. The average difference was 65m (SD=30). The coefficient of concordance between Stratus and Cirrus CMT measurements was rho=0,749 with a high precision and a moderate accuracy. The equation of the line of regression between Stratus and meanCirrus is given by the following: M_stratus = 0,848 x m_cirrus - 4,496 (1).Conclusions: The Cirrus macular cube protocol allows reproducible CMT measurements in patients with active exudative AMD. In cases of upgrading from TD to SD use and vice versa, there is the possibility to predict the measurements by using the equation (1). These real life data and conclusions can help in improving our clinical management of patients with neovascular AMD.

Intracellular trafficking of interleukin-1 receptor I requires Tollip.

Interleukin-1 receptor (IL-1RI) is a master regulator of inflammation and innate immunity. When triggered by IL-1beta, IL-1RI aggregates with IL-1R-associated protein (IL-1RAcP) and forms a membrane proximal signalosome that potently activates downstream signaling cascades. IL-1beta also rapidly triggers endocytosis of IL-1RI. Although internalization of IL-1RI significantly impacts signaling, very little is known about trafficking of IL-1RI and therefore about precisely how endocytosis modulates the overall cellular response to IL-1beta. Upon internalization, activated receptors are often sorted through endosomes and delivered to lysosomes for degradation. This is a highly regulated process that requires ubiquitination of cargo proteins as well as protein-sorting complexes that specifically recognize ubiquitinated cargo. Here, we show that IL-1beta induces ubiquitination of IL-1RI and that via these attached ubiquitin groups, IL-1RI interacts with the ubiquitin-binding protein Tollip. By using an assay to follow trafficking of IL-1RI from the cell surface to late endosomes and lysosomes, we demonstrate that Tollip is required for sorting of IL-1RI at late endosomes. In Tollip-deficient cells and cells expressing only mutated Tollip (incapable of binding IL-1RI and ubiquitin), IL-1RI accumulates on late endosomes and is not efficiently degraded. Furthermore, we show that IL-1RI interacts with Tom1, an ubiquitin-, clathrin-, and Tollip-binding protein, and that Tom1 knockdown also results in the accumulation of IL-1RI at late endosomes. Our findings suggest that Tollip functions as an endosomal adaptor linking IL-1RI, via Tom1, to the endosomal degradation machinery.

Insights into the biogeographical history of the Lower Guinea Forest Domain: evidence for the role of refugia in the intraspecific differentiation of Aucoumea klaineana.

Determining the biogeographical histories of rainforests is central to our understanding of the present distribution of tropical biodiversity. Ice age fragmentation of central African rainforests strongly influenced species distributions. Elevated areas characterized by higher species richness and endemism have been postulated to be Pleistocene forest refugia. However, it is often difficult to separate the effects of history and of present-day ecological conditions on diversity patterns at the interspecific level. Intraspecific genetic variation could yield new insights into history, because refugia hypotheses predict patterns not expected on the basis of contemporary environmental dynamics. Here, we test geographically explicit hypotheses of vicariance associated with the presence of putative refugia and provide clues about their location. We intensively sampled populations of Aucoumea klaineana, a forest tree sensitive to forest fragmentation, throughout its geographical range. Characterizing variation at 10 nuclear microsatellite loci, we were able to obtain phylogeographic data of unprecedented detail for this region. Using Bayesian clustering approaches, we demonstrated the presence of four differentiated genetic units. Their distribution matched that of forest refugia postulated from patterns of species richness and endemism. Our data also show differences in diversity dynamics at leading and trailing edges of the species' shifting distribution. Our results confirm predictions based on refugia hypotheses and cannot be explained on the basis of present-day ecological conditions.