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1.  Revisiting and Revising the Purinosome 
Molecular bioSystems  2014;10(3):369-374.
Some metabolic pathway enzymes are known to organize into multi-enzyme complexes for reasons of catalytic efficiency, metabolite channeling, and other advantages of compartmentalization. It has long been an appealing prospect that de novo purine biosynthesis enzymes form such a complex, termed the “purinosome.” Early work characterizing these enzymes garnered scarce but encouraging evidence for its existence. Recent investigations led to the discovery in human cell lines of purinosome bodies—cytoplasmic puncta containing transfected purine biosynthesis enzymes, which were argued to correspond to purinosomes. New discoveries challenge both the functional and physiological relevance of these bodies in favor of protein aggregation.
PMCID: PMC4151177  PMID: 24413256
2.  Temporal Maps in Appetitive Pavlovian Conditioning 
Behavioural processes  2013;101:15-22.
Previous research suggests animals may integrate temporal information into mental representations, or temporal maps. We examined the parameters under which animals integrate temporal information in three appetitive conditioning experiments. In Experiment 1 the temporal relationship between 2 auditory cues was established during sensory preconditioning (SPC). Subsequently, rats were given first order conditioning (FOC) with one of the cues. Results showed integration of the order of cues between the SPC and FOC training phases. In subsequent experiments we tested the hypothesis that quantitative temporal information can be integrated across phases. In Experiment 2, SPC of two short auditory cues superimposed on a longer auditory cue was followed by FOC of either one of the short cues, or of the long cue at different times in the cue. Contrary to our predictions we did not find evidence of integration of temporal information across the phases of the experiment and instead responding to the SPC cues in Experiment 2 appeared to be dominated by generalization from the FOC cues. In Experiment 3 shorter auditory cues were superimposed on a longer duration light cue but with asynchronous onset and offset of the superimposed cues. There is some evidence consistent with the hypothesis that quantitative discrimination of whether reward should be expected during the early or later parts of a cue could be integrated across experiences. However, the pattern of responding within cues was not indicative of integration of quantitative temporal information. Generalization of expected times of reward during FOC seems to be the dominant determinant of within-cue response patterns in these experiments. Consequently, while we clearly demonstrated the integration of temporal order in the modulation of this dominant pattern we did not find strong evidence of integration of precise quantitative temporal information.
PMCID: PMC3943977  PMID: 24021946
temporal integration; associative learning; temporal map
3.  Dynamic Reorganization of Metabolic Enzymes into Intracellular Bodies 
Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci—such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis—to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality—and dysfunctionality—of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable.
PMCID: PMC4089986  PMID: 23057741
self-assembly; allosteric regulation; fibers; foci; storage bodies; aggregates; metabolic efficiency
4.  Transiently Transfected Purine Biosynthetic Enzymes Form Stress Bodies 
PLoS ONE  2013;8(2):e56203.
It has been hypothesized that components of enzymatic pathways might organize into intracellular assemblies to improve their catalytic efficiency or lead to coordinate regulation. Accordingly, de novo purine biosynthesis enzymes may form a purinosome in the absence of purines, and a punctate intracellular body has been identified as the purinosome. We investigated the mechanism by which human de novo purine biosynthetic enzymes might be organized into purinosomes, especially under differing cellular conditions. Irregardless of the activity of bodies formed by endogenous enzymes, we demonstrate that intracellular bodies formed by transiently transfected, fluorescently tagged human purine biosynthesis proteins are best explained as protein aggregation.
PMCID: PMC3566086  PMID: 23405267
5.  Identification of a therapeutic strategy targeting amplified FGF19 in liver cancer by oncogenomic screening 
Cancer cell  2011;19(3):347-358.
We screened 124 genes that are amplified in human HCC using a mouse hepatoblast model and identified 18 tumor-promoting genes, including CCND1 and its neighbor on 11q13.3, FGF19. Although it is widely assumed that CCND1 is the main driving oncogene of this common amplicon (15% frequency in HCC), both forward-transformation assays and RNAi-mediated inhibition in human HCC cells established that FGF19 is an equally important driver gene in HCC. Furthermore, clonal growth and tumorigenicity of HCC cells harboring the 11q13.3 amplicon were selectively inhibited by RNAi-mediated knockdown of CCND1 or FGF19, as well as by an anti-FGF19 antibody. These results show that 11q13.3 amplification could be an effective biomarker for patients most likely to respond to anti-FGF19 therapy.
PMCID: PMC3061399  PMID: 21397858
6.  Human Cell Chips: Adapting DNA Microarray Spotting Technology to Cell-Based Imaging Assays 
PLoS ONE  2009;4(10):e7088.
Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR), subcellular localization (nuclear translocation of NFκB) and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases) in response to treatment by several chemical effectors (anisomycin, TNFα, and interferon), and we demonstrate scalability by printing a chip with ∼4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability.
PMCID: PMC2760726  PMID: 19862318
7.  Whole genome association analysis shows that ACE is a risk factor for Alzheimer's disease and fails to replicate most candidates from Meta-analysis 
For late onset Alzheimer's disease (LOAD), the only confirmed, genetic association is with the apolipoprotein E (APOE) locus on chromosome 19. Meta-analysis is often employed to sort the true associations from the false positives. LOAD research has the advantage of a continuously updated meta-analysis of candidate gene association studies in the web-based AlzGene database. The top 30 AlzGene loci on May 1st, 2007 were investigated in our whole genome association data set consisting of 1411 LOAD cases and neuropathoiogicaiiy verified controls genotyped at 312,316 SNPs using the Affymetrix 500K Mapping Platform. Of the 30 “top AlzGenes", 32 SNPs in 24 genes had odds ratios (OR) whose 95% confidence intervals that did not include 1. Of these 32 SNPs, six were part of the Affymetrix 500K Mapping panel and another ten had proxies on the Affymetrix array that had >80% power to detect an association with α=0.001. Two of these 16 SNPs showed significant association with LOAD in our sample series. One was rs4420638 at the APOE locus (uncorrected p-value=4.58E-37) and the other was rs4293, located in the angiotensin converting enzyme (ACE) locus (uncorrected p-value=0.014). Since this result was nominally significant, but did not survive multiple testing correction for 16 independent tests, this association at rs4293 was verified in a geographically distinct German cohort (p-value=0.03). We present the results of our ACE replication aiongwith a discussion of the statistical limitations of multiple test corrections in whole genome studies.
PMCID: PMC3076748  PMID: 21537449
Late-onset Alzheimer disease; single nucleotide polymorphism; genome-wide association study; meta-analysis; ACE
8.  GAB2 Alleles Modify Alzheimer’s Risk in APOE ε4 Carriers 
Neuron  2007;54(5):713-720.
The apolipoprotein E (APOE) ε4 allele is the best established genetic risk factor for late-onset Alzheimer’s disease (LOAD). We conducted genome-wide surveys of 502,627 single-nucleotide polymorphisms (SNPs) to characterize and confirm other LOAD susceptibility genes. In ε4 carriers from neuropathologically verified discovery, neuropathologically verified replication, and clinically characterized replication cohorts of 1411 cases and controls, LOAD was associated with six SNPs from the GRB-associated binding protein 2 (GAB2) gene and a common haplotype encompassing the entire GAB2 gene. SNP rs2373115 (p = 9 × 10−11) was associated with an odds ratio of 4.06 (confidence interval 2.81–14.69), which interacts with APOE ε4 to further modify risk. GAB2 was overexpressed in pathologically vulnerable neurons; the Gab2 protein was detected in neurons, tangle-bearing neurons, and dystrophic neuritis; and interference with GAB2 gene expression increased tau phosphorylation. Our findings suggest that GAB2 modifies LOAD risk in APOE ε4 carriers and influences Alzheimer’s neuropathology.
PMCID: PMC2587162  PMID: 17553421

Results 1-8 (8)