Biomedical subjects

James P Brody

Publications and source records attributed to James P Brody.

11 recordsLinked to original sources

PubMed2005

Single-molecule enzymology of chymotrypsin using water-in-oil emulsion.

Single-molecule studies allow the study of subtle activity differences due to local folding in proteins, but are time consuming and difficult because only a few molecules are observed in one experiment. We developed an assay where we can simultaneously measure the activity of hundreds of individual molecules. The assay utilizes a synthetic chymotrypsin substrate that is nonfluorescent before cleavage by chymotrypsin, but is intensely fluorescent afterward. We encapsulated the enzyme and substrate in micron-sized droplets of water surrounded by silicone oil where each microdroplet contains <1 enzyme on average. A microscope and charge-coupled device camera are used to measure the fluorescence intensity of the same individual droplet over time. Based on these measurements, we conclude that enzymatic reactions could occur within this emulsion system, the statistical average activity of individual chymotrypsin molecules is similar to that measured in bulk, and the activity of individual chymotrypsin is heterogeneous.

Animals↗

PubMed2005

Diversity in the activity of individual enzymes.

Although the structure of an enzyme is often depicted as static, it is dynamic. Hence, a population of chemically identical enzymes has not one, but a distribution of structures at any moment in time. Does this have an effect on the activity of the enzyme? This article reviews experiments designed to test the hypothesis that this distribution of structures results in a distribution of enzyme activities. The experiments reviewed here use different enzymes, falvin adenine dinucleotide, beta-galactosidase, alkaline phosphatase, exonuclease I, lactate dehydrogenase I, alpha-chymotrypsin, the 20S proteasome, and horseradish peroxidase. All experiments come to the same conclusion, when measured individually, apparently identical enzymes show a distribution in rates of activity.

Enzymes↗

PubMed2004

Surface plasmon resonance-based sensors to identify cis-regulatory elements.

In eukaryotes, transcription is regulated by multiprotein complexes binding to specific regions of genomic DNA, called cis-regulatory elements. Comprehensive identification of these elements is an important goal of functional genomics. Hence, it is of practical interest to develop a high-throughput assay to identify cis-regulatory elements. Toward that goal, we demonstrate that a surface plasmon resonance-based assay can identify whether a specific region of DNA binds to proteins present in raw nuclear lysate. Specifically, we immobilized a 16-basepair double-stranded DNA region of the SQSTM1 promoter to the Texas Instruments Spreeta, a surface plasmon resonance sensor. As a control, in a separate experiment, we immobilized a similar piece of DNA that differed by only a single base pair. We observed a significant difference in surface plasmon resonance signal when these two probes were exposed to raw nuclear lysate from NIH/3T3 cells. Using a luciferase-reporter vector transfected into live NIH/3T3 cells, we measured a significant difference in transcriptional activity between the two pieces of DNA. We conclude that a surface plasmon resonance-based sensor is capable of identifying physiologically significant cis-regulatory elements.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Electronic manipulation of DNA, proteins, and nanoparticles for potential circuit assembly.

Using gold electrodes lithographically fabricated onto microscope cover slips, DNA and proteins are interrogated both optically (through fluorescence) and electronically (through conductance measurements). Dielectrophoresis is used to position the DNA and proteins at well-defined positions on a chip. Quadrupole electrode geometries are investigated with gaps ranging from 3 to 100 microm; field strengths are typically 10(6) V/m. Twenty nanometer latex beads are also manipulated. The electrical resistance of the electronically manipulated DNA and proteins is measured to be larger than 40 MOhms under the experimental conditions used. The technique of simultaneously measuring resistance while using dielectrophoresis to trap nanoscale objects should find broad applicability.

Coated Materials, Biocompatible↗

PubMed2004

Manipulating nanoparticles in solution with electrically contacted nanotubes using dielectrophoresis.

Dielectrophoresis is an electronic analogue1,2 of optical tweezers3 based on the same physical principle: an ac electric field induces a dipole moment on an object in solution, which then experiences a force proportional to the gradient of the field intensity. For both types of tweezers, this force must compete with thermal Brownian4 motion to be effective, which becomes increasingly difficult as the particle size approaches the nanometer scale. Here we show that this restriction can be overcome by using the large electric field gradient in the vicinity of a carbon nanotube to electronically manipulate nanoparticles down to 2 nm in diameter.

Journal Article↗

PubMed2004

Post-translationally modified S12, absent in transformed breast epithelial cells, is not associated with the 26S proteasome and is induced by proteasome inhibitor.

The 26S proteasome, consisting of the 20S core and 19S regulatory complexes, regulates intracellular protein concentration through proteolytic degradation of targeted substrates. Composition of the 19S regulatory complex as well as posttranslational modifications of the 19S subunits can effectively regulate the activity of the 26S proteasome. Aberrant activity of the 26S proteasome affects the cell cycle, apoptosis and other cellular processes related to cancer. Recent data show an additional proteasome-independent role of 19S subunits in transcriptional regulation. S12 (Rpn8), the human homologue of mouse Mov-34, is a non-ATPase 19S regulatory subunit of the 26S proteasome. Previous studies have identified phosphorylated S12. In our study, we identify a modified S12 isoform (S12-M) with distinct biochemical properties. The S12-M isoform was found in 6 normal, but not 4 transformed, breast epithelial cell lines. Modification of S12 protein can be induced in vitro by addition of the proteasome inhibitor PSI. Modified and unmodified S12 have similar mass, but different isoelectric points, consistent with phosphorylation. In normal cells, unmodified S12 associates with the 26S proteasome, while modified S12-M does not. Whereas transformed cell line nuclei contain neither S12 isoform, S12-M is predominantly cytosolic in normal cells, with the unmodified S12 present in both the nuclei and cytosol. Together with the role of 19S subunits in transcriptional regulation, homology between S12 and eIF3 and TFIIH subunits, coelution with immunoproteasome subunits, and differential posttranslational modification and nuclear localization, these data suggest a differential nuclear function of modified and unmodified S12 in cancer.

Amino Acid Sequence↗

PubMed2004

Identification of the protein Zibra, its genomic organization, regulation, and expression in breast cancer cells.

The mRNA that encodes zibra (zinc, in-between-ring finger, ubiquitin-associated domain), previously known as hypothetical protein FLJ10111, or RNF31 is expressed in several distinct cancers. Little is known about the genomic organization, expression, or regulation of zibra. Using RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we cloned the full-length zibra cDNA from a transformed breast cell line. We identified a novel exon, the 5' untranslated region including the +1 start site, and three alternatively spliced zibra transcripts. The zibra protein contains three zinc ring-finger motifs, an ubiquitin-associated domain, and an in-between-ring-finger domain, characteristic of ubiquitin ligases. We obtained an antibody to zibra and confirmed the presence of translated zibra protein for the first time. Promoter studies localized a core element responsible for basal activity to a 14-bp region in the 5' untranslated region. Although there are numerous consensus Ets factor binding sites in the zibra promoter, we found no affect on promoter activity from Ets-1, PDEF, or PEA-3/E1A-F. Treatment of cells with the proteasome inhibitor I (PSI) decreased zibra protein to an undetectable level after 8 h. Zibra remained undetectable even after 32 h, while mRNA levels remained essentially unchanged. In conclusion, zibra is a translationally regulated putative ubiquitin ligase that is frequently overexpressed in different forms of cancer.

5' Untranslated Regions↗

PubMed2003

p62 overexpression in breast tumors and regulation by prostate-derived Ets factor in breast cancer cells.

p62 is a multifunctional cytoplasmic protein able to noncovalently bind ubiquitin and several signaling proteins, suggesting a regulatory role connected to the ubiquitin-proteasome pathway. No studies to date have linked p62 protein expression with pathological states. Here we demonstrate the overabundance of p62 protein in malignant breast tissue relative to normal breast tissue. The proteasome inhibitor PSI increased p62 mRNA and protein; however, PSI treatment of breast epithelial cells transfected with the p62 promoter did not affect promoter activity. High levels of prostate-derived Ets factor (PDEF) mRNA have been identified in breast cancer compared to normal breast. Only the PSA and maspin promoters have been identified as targets of this transcription factor. Here we show that PDEF stimulates the p62 promoter through at least two sites, and likely acts as a coactivator. PSI treatment abrogates the PDEF-stimulated increase of p62 promoter activity by 50%. Thus, multiple mechanisms for the induction of p62 exist. We conclude that (1) p62 protein is overexpressed in breast cancer; (2) p62 mRNA and protein increase in response to PSI, with no change of basal promoter activity; (3) PDEF upregulates p62 promoter activity through at least two sites; and (4) PSI downregulates PDEF-induced p62 promoter activation through one of these sites.

Acetylcysteine↗

PubMed2002

Significance and statistical errors in the analysis of DNA microarray data.

DNA microarrays are important devices for high throughput measurements of gene expression, but no rational foundation has been established for understanding the sources of within-chip statistical error. We designed a specialized chip and protocol to investigate the distribution and magnitude of within-chip errors and discovered that, as expected from theoretical expectations, measurement errors follow a Lorentzian-like distribution, which explains the widely observed but unexplained ill-reproducibility in microarray data. Using this specially designed chip, we examined a data set of repeated measurements to extract estimates of the distribution and magnitude of statistical errors in DNA microarray measurements. Using the common "ratio of medians" method, we find that the measurements follow a Lorentzian-like distribution, which is problematic for subsequent analysis. We show that a method of analysis dubbed "median of ratios" yields a more Gaussian-like distribution of errors. Finally, we show that the bootstrap algorithm can be used to extract the best estimates of the error in the measurement. Quantifying the statistical error in such measurements has important applications for estimating significance levels, clustering algorithms, and process optimization.

Algorithms↗

PubMed2002

Microarray optimizations: increasing spot accuracy and automated identification of true microarray signals.

In this paper, fluorescent microarray images and various analysis techniques are described to improve the microarray data acquisition processes. Signal intensities produced by rarely expressed genes are initially correctly detected, but they are often lost in corrections for background, log or ratio. Our analyses indicate that a simple correlation between the mean and median signal intensities may be the best way to eliminate inaccurate microarray signals. Unlike traditional quality control methods, the low intensity signals are retained and inaccurate signals are eliminated in this mean and median correlation. With larger amounts of microarray data being generated, it becomes increasingly more difficult to analyze data on a visual basis. Our method allows for the automatic quantitative determination of accurate and reliable signals, which can then be used for normalization. We found that a mean to median correlation of 85% or higher not only retains more data than current methods, but the retained data is more accurate than traditional thresholds or common spot flagging algorithms. We have also found that by using pin microtapping and microvibrations, we can control spot quality independent from initial PCR volume.

Animals↗

PubMed2002

Identification and confirmation of a module of coexpressed genes.

We synthesize a large gene expression data set using dbEST and UniGene. We use guilt-by-association (GBA) to analyze this data set and identify coexpressed genes. One module, or group of genes, was found to be coexpressed mainly in tissue extracted from breast and ovarian cancers, but also found in tissue from lung cancers, brain cancers, and bone marrow. This module contains at least six members that are believed to be involved in either transcritional regulation (PDEF, H2AFO, NUCKS) or the ubiquitin proteasome pathway (PSMD7, SQSTM1, FLJ10111). We confirm these observations of coexpression by real-time RT-PCR analysis of mRNA extracted from four model breast epithelial cell lines.

Adult↗