An essential function of the extreme C-terminus of MDM2 can be provided by MDMX

MDM2 (HDM2) is a ubiquitin ligase that can target the p53 tumor suppressor protein for degradation. The RING domain is essential for the E3 activity of MDM2, and we show here that the extreme C-terminal tail of MDM2 is also critical for efficient E3 activity. Loss of E3 function in MDM2 mutants deleted of the C-terminal tail correlated with a failure of these mutants to oligomerize with MDM2, or with the related protein MDMX (HDMX). However, MDM2 containing point mutations within the C-terminus that inactivated E3 function retained the ability to oligomerize with the wild-type MDM2 RING domain and MDMX, and our results indicate that oligomers containing both wild-type MDM2 and a C-terminal mutant protein retain E3 function both in auto-degradation and degradation of p53. Interestingly, the E3 activity of C-terminal point mutants of MDM2 can also be supported by interaction with wild-type MDMX, suggesting that MDMX can directly contribute to E3 function.     

Effects of Food Availability on Space and Refuge Use by a Neotropical Scatterhoarding Rodent

Animals that rely on refuges for safety can theoretically increase their foraging area without simultaneously increasing predation risk and travel costs by using more refuges. The key prediction of this theory, a negative correlation between food abundance, home range size and the number of refuges used, has never been empirically tested. We determined how home range size and refuge use by the Central American agouti (Dasyprocta punctata) varied across a gradient of abundance of the agoutis' principal food source: seeds and fruits of the palm Astrocaryum standleyanum. We used both manual and automated radio telemetry to measure space use of 11 agoutis during 2 mo of the Astrocaryum fruiting season, and of another set of 10 agoutis during 6 mo in which the animals largely relied on cached Astrocaryum seeds. We found that agoutis living in areas of lower food density had larger home ranges, and that all individuals used multiple refuges. The number of refuges, however, was not correlated with home range size. Consequently, agoutis that had larger home ranges roamed farther from their refuges. These results suggest that agoutis increase their home range size in response to food scarcity at the cost of their safety.     

Terpenoid metabolism in wild-type and transgenic Arabidopsis plants

Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40- to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae, the FaNES1-expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants.     

Cell-cell junction formation: The role of Rap1 and Rap1 guanine nucleotide exchange factors

Rap proteins are Ras-like small GTP-binding proteins that amongst others are involved in the control of cell-cell and cell-matrix adhesion. Several Rap guanine nucleotide exchange factors (RapGEFs) function to activate Rap. These multi-domain proteins, which include C3G, Epacs, PDZ-GEFs, RapGRPs and DOCK4, are regulated by various different stimuli and may function at different levels in junction formation. Downstream of Rap, a number of effector proteins have been implicated in junctional control, most notably the adaptor proteins AF6 and KRIT/CCM1. In this review, we will highlight the latest findings on the Rap signaling network in the control of epithelial and endothelial cell-cell junctions.     

Nuclear PtdIns5P as a transducer of stress signaling: an in vivo role for PIP4Kbeta

Inhibitor of growth protein-2 (ING2) is a nuclear adaptor protein that can regulate p53 and histone acetylation in response to cellular stress and contains a PHD (plant homeodomain) finger that can interact with phosphatidylinositol-5-phosphate (PtdIns5P). However, whether or how nuclear PtdIns5P levels are regulated in response to cellular stress or whether ING2 can sense these changes has not been demonstrated. We show that UV irradiation increases nuclear PtdIns5P levels via inhibition of the activity of the beta isoform of PtdIns5P 4-kinase (PIP4Kbeta), an enzyme that can phosphorylate and remove PtdIns5P. Inhibition of PIP4Kbeta activity occurs through the direct phosphorylation of PIP4Kbeta at Ser326 by the p38 stress-activated protein kinase. Finally, we show that changes in nuclear PtdIns5P are translated into changes in the association of ING2 with chromatin. Our data define a pathway connecting cellular stressors with changes in nuclear PtdIns5P levels and the regulation of PHD motif-containing proteins.     

Prey refuges as predator hotspots: ocelot (Leopardus pardalis) attraction to agouti (Dasyprocta punctata) dens

We tested the hypothesis that prey refuges attract predators, leading to elevated predator activity in the vicinity of refuges. We used camera traps to determine whether the spatial activity of a predator, the ocelot (Leopardus pardalis), was biased toward refuge locations of its principal prey, the agouti (Dasyprocta punctata). We radio-tracked agoutis at night to locate active refuges and compared the activity of ocelots between these refuges and surrounding control grid locations. We found that ocelots visited the area near agouti refuges significantly more often and for longer periods of time than control locations, and that they actively investigated the refuge entrances. Both occupied and unoccupied refuges were visited, but the duration of inspection was longer at occupied refuges. As the ocelots could probably not see the agoutis within the refuges, olfaction likely cued foraging ocelots. Two refuges were repeatedly visited by the same ocelots on different days, suggesting spatial memory. Overall, our results suggest that predators can be attracted to prey refuges or refuging prey. The benefits to prey of staying nearby a refuge would thus be counterbalanced by higher likelihoods of predator encounter. This should stimulate prey to use multiple refuges alternatingly and to not enter or exit refuges at times of high predator activity.     

A critical role for linker DNA in higher-order folding of chromatin fibers

Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.     

Epac1 and PDZ-GEF cooperate in Rap1 mediated endothelial junction control

Epac1 and its effector Rap1 are important mediators of cAMP induced tightening of endothelial junctions and consequential increased barrier function. We have investigated the involvement of Rap1 signalling in basal, unstimulated, barrier function of a confluent monolayer of HUVEC using real time Electric Cell-substrate Impedance Sensing. Depletion of Rap1, but not Epac1, results in a strong decrease in barrier function. This decrease is also observed when cells are depleted of the cAMP independent Rap exchange factors PDZ-GEF1 and 2, showing that PDZ-GEFs are responsible for Rap1 activity in control of basal barrier function. Monolayers of cells depleted of PDZ-GEF or Rap1 show an irregular, zipper-like organization of VE-cadherin and live imaging of VE-cadherin-GFP reveals enhanced junction motility upon depletion of PDZ-GEF or Rap1. Importantly, activation of Epac1 increases the formation of cortical actin bundles at the cell–cell junctions, inhibits junction motility and restores barrier function of PDZ-GEFs depleted, but not Rap1 depleted cells. We conclude that PDZ-GEF activates Rap1 under resting conditions to stabilize cell–cell junctions and maintain basal integrity. Activation of Rap1 by cAMP/Epac1 induces junctional actin to further tighten cell–cell contacts.     

Integrated miRNA and mRNA Expression Profiling in Inflamed Colon of Patients with Ulcerative Colitis

Background

Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA.

Methodology

Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays.

Results

When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression.

Conclusion

Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.