Distinct Mechanisms for Induction and Tolerance Regulate the Immediate Early Genes Encoding Interleukin 1β and Tumor Necrosis Factor α

Interleukin-1β and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPβ and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators. © 2013 Adamik et al

Observation and study of baryonic B decays: B -> D(*) p pbar, D(*) p pbar pi, and D(*) p pbar pi pi

We present a study of ten B-meson decays to a D(*), a proton-antiproton pair, and a system of up to two pions using BaBar's data set of 455x10^6 BBbar pairs. Four of the modes (B0bar -> D0 p anti-p, B0bar -> D*0 p anti-p, B0bar -> D+ p anti-p pi-, B0bar -> D*+ p anti-p pi-) are studied with improved statistics compared to previous measurements; six of the modes (B- -> D0 p anti-p pi-, B- -> D*0 p anti-p pi-, B0bar -> D0 p anti-p pi- pi+, B0bar -> D*0 p anti-p pi- pi+, B- -> D+ p anti-p pi- pi-, B- -> D*+ p anti-p pi- pi-) are first observations. The branching fractions for 3- and 5-body decays are suppressed compared to 4-body decays. Kinematic distributions for 3-body decays show non-overlapping threshold enhancements in m(p anti-p) and m(D(*)0 p) in the Dalitz plots. For 4-body decays, m(p pi-) mass projections show a narrow peak with mass and full width of (1497.4 +- 3.0 +- 0.9) MeV/c2, and (47 +- 12 +- 4) MeV/c2, respectively, where the first (second) errors are statistical (systematic). For 5-body decays, mass projections are similar to phase space expectations. All results are preliminary.Comment: 28 pages, 90 postscript figures, submitted to LP0

A Search for Dark Higgs Bosons

Recent astrophysical and terrestrial experiments have motivated the proposal of a dark sector with GeV-scale gauge boson force carriers and new Higgs bosons. We present a search for a dark Higgs boson using 516 fb-1 of data collected with the BABAR detector. We do not observe a significant signal and we set 90% confidence level upper limits on the product of the Standard Model-dark sector mixing angle and the dark sector coupling constant.Comment: 7 pages, 5 postscript figures, published version with improved plots for b/w printin

The Extended Cleavage Specificity of Human Thrombin

Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1′-Ser/Ala/Gly/Thr, P2′-not acidic and P3′-Arg. Our analysis also identifies an important role for a P3′-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3′-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200–400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1–30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times

In vivo and ex vivo effects of propofol on myocardial performance in rats with obstructive jaundice

BACKGROUND: Responsiveness of the 'jaundiced heart' to propofol is not completely understood. The purpose of this study was to evaluate the effect of propofol on myocardial performance in rats with obstructive jaundice. METHODS: Male Sprague-Dawley rats (n = 40) were randomly allocated into two groups, twenty underwent bile duct ligation (BDL), and 20 underwent a sham operation. Seven days after the surgery, propofol was administered in vivo and ex vivo (Langendorff preparations). Heart rate, left ventricular end-systolic pressure (LVESP) left ventricular end-diastolic pressure (LVEDP), and maximal rate for left ventricular pressure rise and decline (+/- dP/dtmax ) were measured to determine the influence of propofol on the cardiac function of rats. RESULTS: Impaired basal cardiac function was observed in the isolated BDL hearts, whereas in vivo indices of basal cardiac function (LVESP and +/- dP/dt) in vivo were significantly higher in rats that underwent BDL compared with controls. With low or intermediate concentrations of propofol, these indices of cardiac function were within the normal physiologic range in both groups, and responsiveness to propofol was unaffected by BDL. When the highest concentration of propofol was administrated, a significant decline in cardiac function was observed in the BDL group. CONCLUSIONS: In rats that underwent BDL, basal cardiac performance was better in vivo and worse ex vivo compared with controls. Low and intermediate concentrations of propofol did not appear to impair cardiac function in rats with obstructive jaundice.published_or_final_versio

Host Alternation Is Necessary to Maintain the Genome Stability of Rift Valley Fever Virus

Arthropod-borne viruses are transmitted among vertebrate hosts by insect vectors. Unusually, Rift Valley fever virus (RVFV) can also be transmitted by direct contacts of animals/humans with infectious tissues. What are the molecular mechanisms and evolutionary events leading to adopt one mode of transmission rather than the other? Viral replication is implied to be different in a vertebrate host and an invertebrate host. The alternating host cycle tends to limit virus evolution by adopting a compromise fitness level for replication in both hosts. To test this hypothesis, we used a cell culture model system to study the evolution of RVFV. We found that freeing RVFV from alternating replication in mammalian and mosquito cells led to large deletions in the NSs gene carrying the virulence factor. Resulting NSs-truncated viruses were able to protect mice from a challenge with a virulent RVFV. Thus, in nature, virulence is likely maintained by continuous alternating passages between vertebrates and insects. Thereby, depending on the mode of transmission adopted, the evolution of RVFV will be of major importance to predict the outcome of outbreaks

Loss of p53 results in protracted electrographic seizures and development of an aggravated epileptic phenotype following status epilepticus

The p53 tumor suppressor is a multifunctional protein, which regulates cell cycle, differentiation, DNA repair and apoptosis. Experimental seizures up-regulate p53 in the brain, and acute seizure-induced neuronal death can be reduced by genetic deletion or pharmacologic inhibition of p53. However, few long-term functional consequences of p53 deficiency have been explored. Here, we investigated the development of epilepsy triggered by status epilepticus in wild-type and p53-deficient mice. Analysis of electroencephalogram (EEG) recordings during status epilepticus induced by intra-amygdala kainic acid (KA) showed that seizures lasted significantly longer in p53-deficient mice compared with wild-type animals. Nevertheless, neuronal death in the hippocampal CA3 subfield and the neocortex was significantly reduced at 72 h in p53-deficient mice. Long-term continuous EEG telemetry recordings after status epilepticus determined that the sum duration of spontaneous seizures was significantly longer in p53-deficient compared with wild-type mice. Hippocampal damage and neuropeptide Y distribution at the end of chronic recordings was found to be similar between p53-deficient and wild-type mice. The present study identifies protracted KA-induced electrographic status as a novel outcome of p53 deficiency and shows that the absence of p53 leads to an exacerbated epileptic phenotype. Accordingly, targeting p53 to protect against status epilepticus or related neurologic insults may be offset by deleterious consequences of reduced p53 function during epileptogenesis or in chronic epilepsy

Experimental Evolution of an Oncolytic Vesicular Stomatitis Virus with Increased Selectivity for p53-Deficient Cells

Experimental evolution has been used for various biotechnological applications including protein and microbial cell engineering, but less commonly in the field of oncolytic virotherapy. Here, we sought to adapt a rapidly evolving RNA virus to cells deficient for the tumor suppressor gene p53, a hallmark of cancer cells. To achieve this goal, we established four independent evolution lines of the vesicular stomatitis virus (VSV) in p53-knockout mouse embryonic fibroblasts (p53−/− MEFs) under conditions favoring the action of natural selection. We found that some evolved viruses showed increased fitness and cytotoxicity in p53−/− cells but not in isogenic p53+/+ cells, indicating gene-specific adaptation. However, full-length sequencing revealed no obvious or previously described genetic changes associated with oncolytic activity. Half-maximal effective dose (EC50) assays in mouse p53-positive colon cancer (CT26) and p53-deficient breast cancer (4T1) cells indicated that the evolved viruses were more effective against 4T1 cells than the parental virus or a reference oncolytic VSV (MΔ51), but showed no increased efficacy against CT26 cells. In vivo assays using 4T1 syngeneic tumor models showed that one of the evolved lines significantly delayed tumor growth compared to mice treated with the parental virus or untreated controls, and was able to induce transient tumor suppression. Our results show that RNA viruses can be specifically adapted typical cancer features such as p53 inactivation, and illustrate the usefulness of experimental evolution for oncolytic virotherapy