Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and cerine residues in the Epstein-Barr Virus lytic switch protein

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon

Adhesion of <i>Pseudomonas fluorescens</i> biofilms to glass, stainless steel and cellulose

Objectives: The adhesion of colloidal probes of stainless steel, glass and cellulose to Pseudomonas fluorescens biofilms was examined using atomic force microscopy (AFM) to allow comparisons between surfaces to which biofilms might adhere. Results: Biofilm was grown on a stainless steel substrate and covered most of the surface after 96 h. AFM approach and retraction curves were obtained when the biofilm was immersed in a tryptone/soy medium. On approach, all the colloidal probes experienced a long non-contact phase more than 100 nm in length, possibly due to the steric repulsion by extracellular polymers from the biofilm and hydrophobic effects. Retraction data showed that the adhesion varied from position to position on the biofilm. The mean value of adhesion of glass to the biofilm (48 ± 7 nN) was the greatest, followed by stainless steel (30 ± 7 nN) and cellulose (7.8 ± 0.4 nN). Conclusion: The method allows understanding of adhesion between the three materials and biofilm, and development of a better strategy to remove the biofilm from these surfaces relevant to different industrial applications

Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium

Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored. © 2013 Mishra et al

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

PMCID: PMC379391

Methylation profiling of Epstein-Barr virus immediate-early gene promoters, BZLF1 and BRLF1 in tumors of epithelial, NK- and B-cell origins

<p>Abstract</p> <p>Background</p> <p>Epstein-Barr virus (EBV) establishes its latency in EBV-associated malignancies, accompanied by occasionally reactivated lytic cycle. Promoter CpG methylation of EBV genome plays an essential role in maintaining viral latency. Two immediate-early (IE) genes, BZLF1 and BRLF1, induce the switch from latent to lytic infection. Studies of methylation-dependent binding of BZLF1 and BRLF1 to EBV promoters have been well reported, but little is known about the methylation status of <it>BZLF1 </it>and <it>BRLF1 </it>promoters (Zp and Rp) in tumor samples.</p> <p>Methods</p> <p>We evaluated the methylation profiles of Zp and Rp by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS), as well as <it>BZLF1 </it>and <it>BRLF1 </it>expression by semiquantitative reverse transcription (RT)-PCR in tumors of epithelial, NK- and B-cell origins.</p> <p>Results</p> <p>We found that both Zp and Rp were hypermethylated in all studied EBV-positive cell lines and tumors of lymphoid (B- or NK cell) or epithelial origin, while unmethylated Zp and Rp alleles were detected in cell lines expressing <it>BZLF1 </it>and <it>BRLF1</it>. Following azacytidine treatment or combined with trichostatin A (TSA), the expression of <it>BZLF1 </it>and <it>BRLF1 </it>was restored along with concomitant promoter demethylation, which subsequently induced the reactivation of early lytic gene <it>BHRF1 </it>and late lytic gene <it>BLLF1</it>.</p> <p>Conclusions</p> <p>Hypermethylation of Zp and Rp mediates the frequent silencing of <it>BZLF1 </it>and <it>BRLF1 </it>in EBV-associated tumors, which could be reactivated by demethylation agent and ultimately initiated the EBV lytic cascade.</p

Non-Invasive In Vivo Imaging of Calcium Signaling in Mice

Rapid and transient elevations of Ca2+ within cellular microdomains play a critical role in the regulation of many signal transduction pathways. Described here is a genetic approach for non-invasive detection of localized Ca2+ concentration ([Ca2+]) rises in live animals using bioluminescence imaging (BLI). Transgenic mice conditionally expressing the Ca2+-sensitive bioluminescent reporter GFP-aequorin targeted to the mitochondrial matrix were studied in several experimental paradigms. Rapid [Ca2+] rises inside the mitochondrial matrix could be readily detected during single-twitch muscle contractions. Whole body patterns of [Ca2+] were monitored in freely moving mice and during epileptic seizures. Furthermore, variations in mitochondrial [Ca2+] correlated to behavioral components of the sleep/wake cycle were observed during prolonged whole body recordings of newborn mice. This non-invasive imaging technique opens new avenues for the analysis of Ca2+ signaling whenever whole body information in freely moving animals is desired, in particular during behavioral and developmental studies

The influence of in-pregnancy smoking cessation programmes on partner quitting and women's social support mobilization: a randomized controlled trial [ISRCTN89131885]

BACKGROUND: Smoking cessation interventions in pregnancy could influence a woman's social behaviour and her partner's smoking behaviour, but this has not been examined in any published randomized trials. METHOD: 918 women smoking at booking for antenatal care were enrolled in a cluster-randomized trial of three interventions: standard care, self-help manual and enhanced stage-based counselling, or self-help manual, enhanced stage-based counselling and use of an interactive computer program. The outcomes were change in social support received by women between booking for maternity care and 30 weeks gestation and 10 days postpartum and reported cessation in the woman's partner at these times. RESULTS: Few pregnant women's partners stopped smoking (4.1% at 30 weeks of gestation and 5.8% at 10 days postpartum) and the probability of quitting did not differ significantly by trial arm. Women's scores on the Inventory of Socially Supportive Behaviors showed a slight decline from booking to 30 weeks gestation, and a slight increase to 10 days postpartum, but these changes did not differ significantly by trial arm. CONCLUSION: The stage-based interventions tested in this trial aimed partly to influence women's mobilization of support and might have influenced partners' quitting, but there was no evidence that they did so. Given that women and their partners often stopped smoking together, future interventions to prevent smoking in pregnant women could encourage both partners to quit together

High-temperature performance of ferritic steels in fireside corrosion regimes: temperature and deposits

The paper reports high temperature resistance of ferritic steels in fireside corrosion regime in terms of temperature and deposits aggressiveness. Four candidate power plant steels: 15Mo3, T22, T23 and T91 were exposed under simulated air-fired combustion environment for 1000 h. The tests were conducted at 600, 650 and 700 °C according to deposit-recoat test method. Post-exposed samples were examined via dimensional metrology (the main route to quantify metal loss), and mass change data were recorded to perform the study of kinetic behavior at elevated temperatures. Microstructural investigations using ESEM-EDX were performed in order to investigate corrosion degradation and thickness of the scales. The ranking of the steels from most to the least damage was 15Mo3 > T22 > T23 > T91 in all three temperatures. The highest rate of corrosion in all temperatures occurred under the screening deposit