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1.  Development of a Novel Nonradiometric Assay for Nucleic Acid Binding to TDP-43 Suitable for High-Throughput Screening Using AlphaScreen® Technology 
Journal of biomolecular screening  2010;15(9):1099-1106.
TAR DNA binding protein 43 (TDP-43) is a nucleic acid binding protein that is associated with the pathology of cystic fibrosis and neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar dementia. We have developed a robust, quantitative, nonradiometric high-throughput assay measuring oligonucleotide binding to TDP-43 using AlphaScreen® technology. Biotinylated single-stranded TAR DNA (bt-TAR-32) and 6 TG repeats (bt-TG6) bound with high affinity to TDP-43, with KD values of 0.75 nM and 0.63 nM, respectively. Both oligonucleotides exhibited slow dissociation rates, with half-lives of 750 min for bt-TAR-32 and 150 min for bt-TG6. The affinities of unlabeled oligonucleotides, as determined by displacement of either bt-TAR-32 or bt-TG6, were consistent with previous reports of nucleic acid interactions with TDP-43, where increasing TG or UG repeats yield greater affinity. A diversity library of 7360 compounds was screened for inhibition of TDP-43 binding to bt-TAR-32, and a series of compounds was discovered with nascent SAR and IC50 values ranging from 100 nM to 10 μM. These compounds may prove to be useful biochemical tools to elucidate the function of TDP-43 and may lead to novel therapeutics for indications where the TDP-43 nucleic acid interaction is causal to the associated pathology.
PMCID: PMC3426361  PMID: 20855563
TDP-43; AlphaScreen; TAR DNA; ALS; cystic fibrosis
2.  Stress-Induced Changes in Sleep in Rodents: Models and Mechanisms 
Psychological stressors have a prominent effect on sleep in general, and rapid eye movement (REM) sleep in particular. Disruptions in sleep are a prominent feature, and potentially even the hallmark, of posttraumatic stress disorder (PTSD) (Ross et al., 1989). Animal models are critical in understanding both the causes and potential treatments of psychiatric disorders. The current review describes a number of studies that have focused on the impact of stress on sleep in rodent models. The studies are also summarized in Table 1, summarizing the effects of stress in 4-hr blocks in both the light and dark phases. Although mild stress procedures have sometimes produced increases in REM sleep, more intense stressors appear to model the human condition by leading to disruptions in sleep, particularly REM sleep. We also discuss work conducted by our group and others looking at conditioning as a factor in the temporal extension of stress-related sleep disruptions. Finally, we attempt to describe the probable neural mechanisms of the sleep disruptions. A complete understanding of the neural correlates of stress-induced sleep alterations may lead to novel treatments for a variety of debilitating sleep disorders.
PMCID: PMC2215737  PMID: 17764741
Amygdala; corticotropin-releasing factor; stress; sleep; REM; PTSD
3.  Reverse Genetics of Escherichia coli Glycerol Kinase Allosteric Regulation and Glucose Control of Glycerol Utilization In Vivo 
Journal of Bacteriology  2001;183(11):3336-3344.
Reverse genetics is used to evaluate the roles in vivo of allosteric regulation of Escherichia coli glycerol kinase by the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIAGlc (formerly known as IIIglc), and by fructose 1,6-bisphosphate. Roles have been postulated for these allosteric effectors in glucose control of both glycerol utilization and expression of the glpK gene. Genetics methods based on homologous recombination are used to place glpK alleles with known specific mutations into the chromosomal context of the glpK gene in three different genetic backgrounds. The alleles encode glycerol kinases with normal catalytic properties and specific alterations of allosteric regulatory properties, as determined by in vitro characterization of the purified enzymes. The E. coli strains with these alleles display the glycerol kinase regulatory phenotypes that are expected on the basis of the in vitro characterizations. Strains with different glpR alleles are used to assess the relationships between allosteric regulation of glycerol kinase and specific repression in glucose control of the expression of the glpK gene. Results of these studies show that glucose control of glycerol utilization and glycerol kinase expression is not affected by the loss of IIAGlc inhibition of glycerol kinase. In contrast, fructose 1,6-bisphosphate inhibition of glycerol kinase is the dominant allosteric control mechanism, and glucose is unable to control glycerol utilization in its absence. Specific repression is not required for glucose control of glycerol utilization, and the relative roles of various mechanisms for glucose control (catabolite repression, specific repression, and inducer exclusion) are different for glycerol utilization than for lactose utilization.
PMCID: PMC99631  PMID: 11344141

Results 1-3 (3)