PubMed Health⌕ Search

Biomedical subjects

Jennifer L Hess

Publications and source records attributed to Jennifer L Hess.

8 recordsLinked to original sources
PubMed2006

Fibrinogen fragment D is necessary and sufficient to anchor a surface plasminogen-activating complex in Streptococcus pyogenes.

In this study, the importance of different domains of the fibrinogen molecule in the binding and assembly of a surface plasminogen (plgn) activator has been analyzed. This was achieved using SELDI technology that enabled dissociation of bound fragments from intact bacteria and accurate distinction between fibrinogen fragments based on their molecular mass. These studies indicate that Streptococcus pyogenes binds directly to human fibrinogen fragment D but not fragment E. The predominant surface proteins binding to fragment D were associated with the mrp gene product. Surface-associated fibrinogen fragment D was capable of anchoring a functional surface plgn activator complex. Taken together, these data indicated that fragment D of fibrinogen is necessary and sufficient to anchor a plgn activator complex on the surface of Streptococcus pyogenes.

Fibrin Fibrinogen Degradation Products↗
PubMed2006

Application of immunoproteomics to rapid cytokine detection.

Cytokines are pivotal to a balanced innate or cell-mediated immune response, and can be indicative of disease progression and/or resolution. Methods to measure key cytokines rapidly with accuracy, precision, and sensitivity are consequently important. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1-3 h) required to carry out the assays. An alternative, semi-quantitative approach described here, uses an immunological capture step and a mass spectral readout. The goal of the assay is speed rather than sensitivity or precision.

Animals↗
PubMed2005

Practical applications of high-affinity, albumin-binding proteins from a group G streptococcal isolate.

Binding proteins that have high affinities for mammalian plasma proteins that are expressed on the surface of bacteria have proven valuable for the purification and detection of several biologically important molecules from human and animal plasma or serum. In this study, we have isolated a high affinity albumin-binding molecule from a group G streptococcal isolate of bovine origin and have demonstrated that the isolated protein can be biotinylated without loss of binding activity and can be used as a tracer for quantification of human serum albumin (HSA). The binding protein can be immobilized and used as a selective capture reagent in a competitive ELISA format using a biotinylated HSA tracer. In this assay format, the sensitivity of detection for 50% inhibition of binding of HSA was less than 1 microg/ml. When attached to the bacterial surface, this binding protein can be used to deplete albumin from human plasma, as analyzed by surface-enhanced laser desorption ionization time of flight mass spectrometry.

Bacterial Proteins↗
PubMed2005

Immunoproteomics.

A novel immunoproteomic assay, combining specificity of antibody with precision of mass spectral analysis is described, and a number of practical applications are presented. The assay is carried out in three steps. The first step of the assay involves antibody immobilization, using a bacterial Fc binding support. The second step is antigen capture and washing to remove non-specific binding. The third step involves analysis of the captured antigens by SELDI-TOF. The assay has many advantages in sensitivity, speed, and economy of reagents in detection of specific antigens or antibodies. In addition, under appropriate experimental conditions, semi-quantitative data may be obtained. By combining the increasing range of selective specific antibody reagents available, in part due to advances in antibody engineering technology, and the resolving power available, using mass spectrometry, immunoproteomics is a valuable technique in proteomic analysis. A number of examples of the application of this technique to analysis of biological systems are presented.

Animals↗
PubMed2004

Telomerase activity measurement in magnetically captured epithelial cells: comparison of slab-gel and capillary electrophoresis.

We have compared telomerase activity measurements by slab-gel and capillary electrophoresis in cultured cells (A549 and H125 human cancer cell lines) and in cells isolated from clinical peripheral blood specimens epithelial cells of patients with lung and esophageal cancer. Telomerase activity was determined using the telomerase repeat amplification protocol (TRAP) assay with phosphoimager scanning of slab-gels and by laser-induced fluorescence capillary electrophoresis (LIF-CE). Experiments using A549 and H125 cells were performed to determine the reproducibility of each method and to identify the contribution of each stage of the TRAP/polymerase chain reaction (PCR) assay to the variability. In these experiments, it was found that more than half of the overall variability (coefficient of variation, CV = 35%) of the slab-gel method and almost all of the overall variability (CV = 20%) of the CE method was due to the PCR stage of the TRAP assay. In the clinical samples, classification as positive or negative was by visual inspection of the slab-gel and CE electropherograms for the presence of the characteristic 6 base-pair TRAP ladder and by GeneScan analysis of the CE. We examined several criteria including the use of 3, 4, or 5 TRAP bands as the definition of a positive test. Using the slab-gel method, the 5-band criterion gave 40% sensitivity with 100% specificity (no false positives in inactive controls). The CE method yielded a comparable 38% sensitivity and 100% specificity using this criterion. These data indicate that detection of telomerase activity in epithelial cells isolated from peripheral blood has a useful level of sensitivity and specificity and may be useful in the detection and monitoring of aerodigestive cancers. However, analysis by slab-gel is cumbersome and the precision is poor (inter-replicate CV = 20%) compared to LIF-CE (CV = 5%). A high-throughput CE-LIF detection platform will be indispensable for validation studies of telomerase activity measurements.

Electrophoresis, Capillary↗
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

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↗