A gastrin transcript expressed in gastrointestinal cancer cells contains an internal ribosome entry site

As the hormone gastrin promotes gastrointestinal (GI) cancer progression by triggering survival pathways, regulation of gastrin expression at the translational level was explored. Sequence within the 5′ untranslated region of a gastrin transcript expressed in GI cancer cells was investigated, then cloned into a bicistronic vector upstream of firefly luciferase and transfected into a series of GI cancer cell lines. Firefly luciferase activity was measured relative to that of a cap-dependent Renilla luciferase. A gastrin transcript that was different from that described in Ensembl was expressed in GI cancer cells. Its transcription appears to be initiated within the region designated as the gene's first intron. In GI cancer cells transfected with the bicistronic construct, firefly luciferase activity increased 8–15-fold compared with the control vector, and there was a further induction of the signal (up to 25-fold) following exposure of the cells to genotoxic stress or hypoxia, suggesting that the sequence acts as an internal ribosome entry site. These data suggest that the gastrin transcript within GI cancer cells contains an internal ribosome entry site that may allow continued expression of gastrin peptides when normal translational mechanisms are inactive, such as in hypoxia, thereby promoting cancer cell survival

A 340/380 nm light emitting diode illuminator for Fura-2 AM ratiometric Ca2+ imaging of live cells with better than 5 nM precision

We report the first demonstration of a fast wavelength-switchable 340/380 nm light emitting diode (LED) illuminator for Fura-2 ratiometric Ca2+ imaging of live cells. The LEDs closely match the excitation peaks of bound and free Fura-2 and enables the precise detection of cytosolic Ca2+ concentrations, which is only limited by the Ca2+ response of Fura-2. Using this illuminator, we have shown that Fura-2 acetoxymethyl ester (AM) concentrations as low as 250 nM can be used to detect induced Ca2+ events in tsA-201 cells and while utilizing the 150 µs switching speeds available, it was possible to image spontaneous Ca2+ transients in hippocampal neurons at a rate of 24.39 Hz that were blunted or absent at typical 0.5 Hz acquisition rates. Overall, the sensitivity and acquisition speeds available using this LED illuminator significantly improves the temporal resolution that can be obtained in comparison to current systems and supports optical imaging of fast Ca2+ events using Fura-2

Asymptotic behaviour of random tridiagonal Markov chains in biological applications

Discrete-time discrete-state random Markov chains with a tridiagonal generator are shown to have a random attractor consisting of singleton subsets, essentially a random path, in the simplex of probability vectors. The proof uses the Hilbert projection metric and the fact that the linear cocycle generated by the Markov chain is a uniformly contractive mapping of the positive cone into itself. The proof does not involve probabilistic properties of the sample path and is thus equally valid in the nonautonomous deterministic context of Markov chains with, say, periodically varying transitions probabilities, in which case the attractor is a periodic path.Comment: 13 pages, 22 bibliography references, submitted to DCDS-B, added references and minor correction

Mitogen-activated protein kinase phosphatase-2 deletion impairs synaptic plasticity and hippocampal-dependent memory

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer’s disease. Thus there is great interest in understanding the signalling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2-/- mice), we show that long-term potentiation (LTP) is impaired in MKP-2-/- mice compared to MKP-2+/+ controls whereas neuronal excitability, evoked synaptic transmission and paired-pulse facilitation remain unaltered. Furthermore, spontaneous excitatory postsynaptic currents (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2-/- mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures which may account for the increase in spontaneous EPSC frequency. In addition no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2-/- mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signalling

Synergistic Impacts of Organic Acids and pH on Growth of Pseudomonas aeruginosa: A Comparison of Parametric and Bayesian Non-parametric Methods to Model Growth

Different weak organic acids have significant potential as topical treatments for wounds infected by opportunistic pathogens that are recalcitrant to standard treatments. These acids have long been used as bacteriostatic compounds in the food industry, and in some cases are already being used in the clinic. The effects of different organic acids vary with pH, concentration, and the specific organic acid used, but no studies to date on any opportunistic pathogens have examined the detailed interactions between these key variables in a controlled and systematic way. We have therefore comprehensively evaluated the effects of several different weak organic acids on growth of the opportunistic pathogen Pseudomonas aeruginosa. We used a semi-automated plate reader to generate growth profiles for two different strains (model laboratory strain PAO1 and clinical isolate PA1054 from a hospital burns unit) in a range of organic acids at different concentrations and pH, with a high level of replication for a total of 162,960 data points. We then compared two different modeling approaches for the interpretation of this time-resolved dataset: parametric logistic regression (with or without a component to include lag phase) vs. non-parametric Gaussian process (GP) regression. Because GP makes no prior assumptions about the nature of the growth, this method proved to be superior in cases where growth did not follow a standard sigmoid functional form, as is common when bacteria grow under stress. Acetic, propionic and butyric acids were all more detrimental to growth than the other acids tested, and although PA1054 grew better than PAO1 under non-stress conditions, this difference largely disappeared as the levels of stress increased. As expected from knowledge of how organic acids behave, their effect was significantly enhanced in combination with low pH, with this interaction being greatest in the case of propionic acid. Our approach lends itself to the characterization of combinatorial interactions between stressors, especially in cases where their impacts on growth render logistic growth models unsuitable

Interaction of eukaryotic translation initiation factor 4G with the nuclear cap-binding complex provides a link between nuclear and cytoplasmic functions of the m7 guanosine cap

In eukaryotes the majority of mRNAs have an m7G cap that is added cotranscriptionally and that plays an important role in many aspects of mRNA metabolism. The nuclear cap-binding complex (CBC; consisting of CBP20 and CBP80) mediates the stimulatory functions of the cap in pre-mRNA splicing, 3' end formation, and U snRNA export. As little is known about how nuclear CBC mediates the effects of the cap in higher eukaryotes, we have characterized proteins that interact with CBC in HeLa cell nuclear extracts as potential mediators of its function. Using cross-linking and coimmunoprecipitation, we show that eukaryotic translation initiation factor 4G (eIF4G), in addition to its function in the cytoplasm, is a nuclear CBC-interacting protein. We demonstrate that eIF4G interacts with CBC in vitro and that, in addition to its cytoplasmic localization, there is a significant nuclear pool of eIF4G in mammalian cells in vivo. Immunoprecipitation experiments suggest that, in contrast to the cytoplasmic pool, much of the nuclear eIF4G is not associated with eIF4E (translation cap binding protein of eIF4F) but is associated with CBC. While eIF4G stably associates with spliceosomes in vitro and shows close association with spliceosomal snRNPs and splicing factors in vivo, depletion studies show that it does not participate directly in the splicing reaction. Taken together the data indicate that nuclear eIF4G may be recruited to pre-mRNAs via its interaction with CBC and accompanies the mRNA to the cytoplasm, facilitating the switching of CBC for eIF4F. This may provide a mechanism to couple nuclear and cytoplasmic functions of the mRNA cap structure

Compartmentalisation and localisation of the translation initiation factor (eIF) 4F complex in normally growing fibroblasts

Previous observations of association of mRNAs and ribosomes with subcellular structures highlight the importance of localised translation. However, little is known regarding associations between eukaryotic translation initiation factors and cellular structures within the cytoplasm of normally growing cells. We have used detergent-based cellular fractionation coupled with immunofluorescence microscopy to investigate the subcellular localisation in NIH3T3 fibroblasts of the initiation factors involved in recruitment of mRNA for translation, focussing on eIF4E, the mRNA cap-binding protein, the scaffold protein eIF4GI and poly(A) binding protein (PABP). We find that these proteins exist mainly in a soluble cytosolic pool, with only a subfraction tightly associated with cellular structures. However, this "associated" fraction was enriched in active "eIF4F" complexes (eIF4E.eIF4G.eIF4A.PABP). Immunofluorescence analysis reveals both a diffuse and a perinuclear distribution of eIF4G, with the perinuclear staining pattern similar to that of the endoplasmic reticulum. eIF4E also shows both a diffuse staining pattern and a tighter perinuclear stain, partly coincident with vimentin intermediate filaments. All three proteins localise to the lamellipodia of migrating cells in close proximity to ribosomes, microtubules, microfilaments and focal adhesions, with eIF4G and eIF4E at the periphery showing a similar staining pattern to the focal adhesion protein vinculin

TAp73 contributes to the oxidative stress response by regulating protein synthesis.

TAp73 is a transcription factor that plays key roles in brain development, aging, and cancer. At the cellular level, TAp73 is a critical homeostasis-maintaining factor, particularly following oxidative stress. Although major studies focused on TAp73 transcriptional activities have indicated a contribution of TAp73 to cellular metabolism, the mechanisms underlying its role in redox homeostasis have not been completely elucidated. Here we show that TAp73 contributes to the oxidative stress response by participating in the control of protein synthesis. Regulation of mRNA translation occupies a central position in cellular homeostasis during the stress response, often by reducing global rates of protein synthesis and promoting translation of specific mRNAs. TAp73 depletion results in aberrant ribosomal RNA (rRNA) processing and impaired protein synthesis. In particular, polysomal profiles show that TAp73 promotes the integration of mRNAs that encode rRNA-processing factors in polysomes, supporting their translation. Concurrently, TAp73 depletion causes increased sensitivity to oxidative stress that correlates with reduced ATP levels, hyperactivation of AMPK, and translational defects. TAp73 is important for maintaining active translation of mitochondrial transcripts in response to oxidative stress, thus promoting mitochondrial activity. Our results indicate that TAp73 contributes to redox homeostasis by affecting the translational machinery, facilitating the translation of specific mitochondrial transcripts. This study identifies a mechanism by which TAp73 contributes to the oxidative stress response and describes a completely unexpected role for TAp73 in regulating protein synthesis

A role for eukaryotic initiation factor 4B overexpression in the pathogenesis of diffuse large B-cell lymphoma.

Dysregulated expression of factors that control protein synthesis is associated with poor prognosis of many cancers, but the underlying mechanisms are not well defined. Analysis of the diffuse large B-cell lymphoma (DLBCL) translatome revealed selective upregulation of mRNAs encoding anti-apoptotic and DNA repair proteins. We show that enhanced synthesis of these proteins in DLBCL is mediated by the relief of repression that is normally imposed by structure in the 5'-untranslated regions of their corresponding mRNAs. This process is driven by signaling through mammalian target of rapamycin, resulting in increased synthesis of eukaryotic initiation factor (eIF) 4B complex (eIF4B), a known activator of the RNA helicase eIF4A. Reducing eIF4B expression alone is sufficient to decrease synthesis of proteins associated with enhanced tumor cell survival, namely DAXX, BCL2 and ERCC5. Importantly, eIF4B-driven expression of these key survival proteins is directly correlated with patient outcome, and eIF4B, DAXX and ERCC5 are identified as novel prognostic markers for poor survival in DLBCL. Our work provides new insights into the mechanisms by which the cancer-promoting translational machinery drives lymphomagenesis