ChIPing the cistrome of PXR in mouse liver

The pregnane X receptor (PXR) is a key regulator of xenobiotic metabolism and disposition in liver. However, little is known about the PXR DNA-binding signatures in vivo, or how PXR regulates novel direct targets on a genome-wide scale. Therefore, we generated a roadmap of hepatic PXR bindings in the entire mouse genome [chromatin immunoprecipitation (ChIP)-Seq]. The most frequent PXR DNA-binding motif is the AGTTCA-like direct repeat with a 4bp spacer [direct repeat (DR)-4)]. Surprisingly, there are also high motif occurrences with spacers of a periodicity of 5 bp, forming a novel DR-(5n + 4) pattern for PXR binding. PXR-binding overlaps with the epigenetic mark for gene activation (histone-H3K4-di-methylation), but not with epigenetic marks for gene suppression (DNA methylation or histone-H3K27-tri-methylation) (ChIP-on-chip). After administering a PXR agonist, changes in mRNA of most PXR-direct target genes correlate with increased PXR binding. Specifically, increased PXR binding triggers the trans-activation of critical drug-metabolizing enzymes and transporters. The mRNA induction of these genes is absent in PXR-null mice. The current work provides the first in vivo evidence of PXR DNA-binding signatures in the mouse genome, paving the path for predicting and further understanding the multifaceted roles of PXR in liver.     

Effects of continuous low dose-rate irradiation: computer simulations

The effects of continuous low dose-rate irradiation are studied with a computer model that incorporates cell kinetics and the accumulation and repair of radiation damage. This theoretical approach independently explores the effects on survival curves of a phase block, inherited damage and proliferation by dying cells. The computer model is a Monte Carlo simulation which follows the evolution in time of the family trees of a growing cell population under continuous irradiation. The model uses as input the measured phase-specific survival curves for acute exposures and the cell kinetic parameters to generate survival curves for continuous low dose-rate irradiations. Cell survival curves for Chinese hamster lung cells (V79) for dose rates ranging from 15 to 500 cGy/h have been generated using various model assumptions. The model shows that for these cells a G2 block will maximize cell killing for an optimum dose rate near 75 cGy/h. The effect on survival curves of inherited damage, as well as that of the proliferation by dying cells, is shown to increase monotonically with decreasing dose rates, and to be quite large at low dose rates.     

Peptide models for the membrane destabilizing actions of viral fusion proteins.

The fusion of enveloped viruses to target membranes is promoted by certain viral fusion proteins. However, many other proteins and peptides stabilize bilayer membranes and inhibit membrane fusion. We have evaluated some characteristics of the interaction of peptides that are models of segments of measles and influenza fusion proteins with membranes. Our results indicate that these models of the fusogenic domains of viral fusion proteins promote conversion of model membrane bilayers to nonbilayer phases. This is opposite to the effects of peptides and proteins that inhibit viral fusion. A peptide model for the fusion segment of the HA protein of influenza increased membrane leakage as well as promoted the formation of nonbilayer phases upon acidification from pH 7-5. We analyze the gross conformational features of the peptides, and speculate on how these conformational features relate to the structures of the intact proteins and to their role in promoting membrane fusion.     

Priority list of research areas for radiological nuclear threat countermeasures

To help the nation prepare for the possibility of a terrorist attack using radiological and nuclear devices, the Office of Science and Technology Policy and the Homeland Security Council established an interagency working group. The working group deliberated on the research needs for radiological/ nuclear threat countermeasures and identified and prioritized 18 areas for further attention. The highest priorities were given to research on (1) radioprotectors for use prior to exposure; (2) therapeutic agents for postexposure treatment; (3) antimicrobial therapy for infections associated with radiation exposure; (4) cytokines and growth factors; (5) mechanisms of radiation injury at the molecular, cellular, tissue and organism levels; and (6) automation of biodosimetric assays. High priority was given to (1) developing biomarkers for biodosimetry; (2) enhancing training in the radiation sciences; (3) exploring the consequences of combined injury; (4) establishing a repository of information regarding investigational countermeasures; and (5) following the health of an exposed population to better prepare for subsequent events. The research areas that the committee felt required the attention of the radiation research community are described in this report in an effort to inform this community about the needs of the nation and to encourage researchers to address these critical issues.     

N-acetylcysteine and celecoxib lessen cadmium cytotoxicity which is associated with cyclooxygenase-2 up-regulation in mouse neuronal cells

In many neurodegenerative disorders, aggregates of ubiquitinated proteins are detected in neuronal inclusions, but their role in neurodegeneration remains to be defined. To identify intracellular mechanisms associated with the appearance of ubiquitin-protein aggregates, mouse neuronal HT4 cells were treated with cadmium. This heavy metal is a potent cell poison that mediates oxidative stress and disrupts the ubiquitin/proteasome pathway. In the current studies, the following intracellular events were found to be also induced by cadmium: (i) a specific rise in cyclooxygenase-2 (COX-2) gene expression but not COX-1; (ii) an increase in the extracellular levels of the proinflammatory prostaglandin E2, a product of COX-2; and (iii) production of 4-hydroxy-2-nonenal-protein adducts, which result from lipid peroxidation. In addition, cadmium treatment led to the accumulation of high molecular weight ubiquitin-COX-2 conjugates and perturbed COX-2 glycosylation. The thiol-reducing antioxidant N-acetylcysteine, and, to a lesser extent, the COX-2 inhibitor celecoxib, attenuated the loss of cell viability induced by cadmium demonstrating that oxidative stress and COX-2 activation contribute to cadmium cytotoxicity. These findings establish that disruption of the ubiquitin/proteasome pathway is not the only event triggered by cadmium. This oxidative stressor also activates COX-2 function. Both events could be triggered by formation of 4-hydroxy-2-nonenal as a result of cadmium-induced lipid peroxidation. Proinflammatory responses stimulated by oxidative stressors that mimic the cadmium effects may, therefore, be important initiators of the neurodegenerative process and exacerbate its progress.     

Synthesis of peptidyl methylcoumarin esters as substrates and active-site titrants for the prohormone processing proteases Kex2 and PC2

Peptidyl methylcoumarin amides are well established as model substrates for understanding protease specificity, but the corresponding methylcoumarin esters have attracted scant attention despite their potential utility in active-site titration and mechanistic characterization. We have devised techniques for the synthesis and deprotection of extended peptidyl methylcoumarin esters in good to moderate yields, and we have demonstrated their suitability for steady-state characterization and active-site titration of the Saccharomyces cerevisiae processing protease Kex2. Additionally, we have used one of these compounds to active-site titrate the homologous enzyme PC2, which had not previously been feasible using other types of substrates. These compounds should thus prove widely suitable for use as substrates and active-site titrants not only for proteases of the prohormone processing family but also for a wide range of other serine proteases.     

AAV-Mediated Gene Delivery in a Feline Model of Sandhoff Disease Corrects Lysosomal Storage in the Central Nervous System

Sandhoff disease (SD) is an autosomal recessive neurodegenerative disease caused by a mutation in the gene for the β-subunit of β-N-acetylhexosaminidase (Hex), resulting in the inability to catabolize ganglioside GM2 within the lysosomes. SD presents with an accumulation of GM2 and its asialo derivative GA2, primarily in the central nervous system. Myelin-enriched glycolipids, cerebrosides and sulfatides, are also decreased in SD corresponding with dysmyelination. At present, no treatment exists for SD. Previous studies have shown the therapeutic benefit of adeno-associated virus (AAV) vector-mediated gene therapy in the treatment of SD in murine and feline models. In this study, we treated presymptomatic SD cats with AAVrh8 vectors expressing feline Hex in the thalamus combined with intracerebroventricular (Thal/ICV) injections. Treated animals showed clearly improved neurologic function and quality of life, manifested in part by prevention or attenuation of whole-body tremors characteristic of untreated animals. Hex activity was significantly elevated, whereas storage of GM2 and GA2 was significantly decreased in tissue samples taken from the cortex, cerebellum, thalamus, and cervical spinal cord. Treatment also increased levels of myelin-enriched cerebrosides and sulfatides in the cortex and thalamus. This study demonstrates the therapeutic potential of AAV for feline SD and suggests a similar potential for human SD patients.