Biomedical subjects

W Edward Highsmith

Publications and source records attributed to W Edward Highsmith.

5 recordsLinked to original sources

PubMed2003

High-throughput analysis of telomerase by capillary electrophoresis.

The enzyme telomerase is expressed in (85-90)% of all human cancers, but not in normal, non-stem cell somatic tissues. Clinical assays for telomerase in easily obtained body fluids would have great utility as noninvasive, cost-effective methods for the early detection of cancer. The most commonly used method for the detection and quantification of telomerase enzyme activity is the polymerase chain reaction (PCR)-based assay known as the telomerase repeat amplification protocol or TRAP assay. Most of the TRAP assay systems use a slab-gel based electrophoresis system to size and quantify the PCR-amplified extension products. We are developing high-throughput capillary electrophoresis (CE) methods for the analysis of TRAP/PCR products. The TRAP assay was conducted on lysates of the human lung cancer cell line A-549 in reactions containing 5-100 cells. TRAP/PCR products were generated using a fluorescent 4,7,2'4'5'7',-hexachloro-6-carboxyfluorescein(HEX)-labeled TS primer and analyzed on the Applied Biosystems Model 310 CE system using POP4 polymer. After analysis with GeneScan and Genotyper software, the total peak areas of the TRAP ladder extension products were computed using Microsoft Excel. Results were compared with unlabeled TRAP/PCR products analyzed on the Bio-Rad BioFocus 3000 CE system using 6% high molecular weight polyvinylpyrrolidone (HMW PVP) polymer and SYBR Green I dye. Both CE systems were able to resolve the TRAP ladder products with high reproducibility and sensitivity (5-15 cells). With the appropriate robotic sample handling system, these CE methods would enable performing the telomerase TRAP assay with increased sensitivity, reproducibility and automation over slab-gel methods.

Base Sequence↗

PubMed2002

Phage display technology: clinical applications and recent innovations.

Phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. Phage display libraries permit the selection of peptides and proteins, including antibodies, with high affinity and specificity for almost any target. A crucial advantage of this technology is the direct link that exists between the experimental phenotype and its encapsulated genotype, which allows the evolution of the selected binders into optimized molecules. Phage display facilitates engineering of antibodies with regard to their size, valency, affinity, and effector functions. The selection of antibodies and peptides from libraries displayed on the surface of filamentous phage has proven significant for routine isolation of peptides and antibodies for diagnostic and therapeutic applications. This review serves as an introduction to phage display, antibody engineering, the development of phage-displayed peptides and antibody fragments into viable diagnostic reagents, and recent trends in display technology.

Animals↗

PubMed2002

Identifying and quantifying sources of variation in microarray data using high-density cDNA membrane arrays.

Microarray experiments involve many steps, including spotting cDNA, extracting RNA, labeling targets, hybridizing, scanning, and analyzing images. Each step introduces variability, confounding our ability to obtain accurate estimates of the biological differences between samples. We ran repeated experiments using high-density cDNA microarray membranes (Research Genetics Human GeneFilters Microarrays Version I) and 33P-labeled targets. Total RNA was extracted from a Burkitt lymphoma cell line (GA-10). We estimated the components of variation coming from: (1) image analysis, (2) exposure time to PhosphorImager screens, (3) differences in membranes, (4) reuse of membranes, and (5) differences in targets prepared from two independent RNA extractions. Variation was assessed qualitatively using a clustering algorithm and quantitatively using a version of ANOVA adapted to multivariate microarray data. The largest contribution to variation came from reusing membranes, which contributed 38% of the total variation. Differences in membranes and in exposure time each contributed about 10%. Differences in target preparations contributed less than 5%. The effect of image quantification was negligible. Much of the effect from reusing membranes was attributable to increasing levels of background radiation and can be reduced by using membranes at most four times. The effects of exposure time, which were partly attributable to variation in the scanning process, can be minimized by using the same exposure time for all experiments.

Algorithms↗

PubMed2002

Telomerase as a diagnostic and therapeutic target for cancer.

Telomerase is a ribonucleoprotein enzyme responsible for the elongation of telomeres at the ends of chromosomes. It is widely expressed in most cancers, while absent from most normal somatic cells. Telomerase is partially responsible for the cellular immortalization that allows human cancers to progress indefinitely. Due to its widespread occurrence in cancer and its crucial role in the maintenance of the tumor, telomerase is an attractive target for cancer diagnosis and treatment.

Animals↗

PubMed2002

Telomerase detection in body fluids.

BACKGROUND: Telomerase is a ribonucleoprotein that maintains chromosomal telomere length. Telomerase is not active in nonmalignant somatic cells, but is activated in most human cancers. Telomerase activity in easily obtainable body fluids that bathe tumors may be a useful cancer marker, especially when used in conjunction with conventional cytology. APPROACH: Results from studies that assayed telomerase activity in easily obtainable body fluids are reviewed. CONTENT: The telomerase repeat amplification protocol (TRAP) assay has been used to measure telomerase activity in body fluids, including ascites, pleural effusions, pelvic washes, bronchial washings, bronchial lavage, urine, bladder washings, oral rinses, and plasma. Telomerase activity has sensitivities of 60-90% as a tumor marker with clinical specificities for cancer of approximately 90%. Telomerase activity is more sensitive than conventional cytology, the sensitivity of which was 40-65% in various studies. SUMMARY: Telomerase activity in body fluids, as measured by the TRAP assay, is a sensitive potential tumor marker that might help increase the cancer detection rate and the cancer treatment success rate when combined with conventional cytology.

Biomarkers, Tumor↗