Are you Richard M. McGowen?

Claim your profile

Publications (4)2.49 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Nuclear factor-kappaB (NF-kappaB) is a ubiquitous redox-sensitive transcription factor involved in the pro-inflammatory response to several factors, including cytokines and oxidative stress. Upon activation, NF-kappaB translocates into the nucleus and binds to specific nucleotide sequences. The cellular responses to inflammatory and stress signals have been implicated in disease conditions, such as atherosclerosis, cancer, diabetes, and Alzheimer's disease. The conventional method for detection of NF-kappaB -DNA binding activity is the electrophoretic mobility shift assay (EMSA), which is time-consuming and non-quantitative. Here, we report (a) development of a rapid, sensitive and quantitative chemiluminescent immunoassay (QCI) for analysis of NF-kappaB DNA-binding activity, and (b) validation of the QCI with the EMSA using nuclear and cytosolic extracts from cultured prostate cancer cells (PC3), rat liver homogenates and human lymphocytes. The QCI for analysis of NF-kappaB DNA binding activity has advantages over the EMSA: (1) Higher speed: 3-5h post sample preparation, (2) Greater sensitivity: 10pg NF-kappaB/well, (3) Quantitative: linear range: 10-1000pg NF-kappaB; r2 = 0.999 (4) High throughput adaptability: 96-well plate format can analyze up to 40 samples in duplicate, (5) Safety: No radioactive isotopes, (6) Simplicity, and (7) Capability of measurement of both activated (free) NF-KB which is translocated into the nucleus and total (bound + unbound) NF-kappaB present in the cytosol/cell.
    Journal of Immunoassay and Immunochemistry 02/2005; 26(2):125-43. · 0.73 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Nuclear factor‐κB (NF‐κB) is a ubiquitous redox‐sensitive transcription factor involved in the pro‐inflammatory response to several factors, including cytokines and oxidative stress. Upon activation, NF‐κB translocates into the nucleus and binds to specific nucleotide sequences. The cellular responses to inflammatory and stress signals have been implicated in disease conditions, such as atherosclerosis, cancer, diabetes, and Alzheimer's disease. The conventional method for detection of NF‐κB ‐DNA binding activity is the electrophoretic mobility shift assay (EMSA), which is time‐consuming and non‐quantitative. Here, we report (a) development of a rapid, sensitive and quantitative chemiluminescent immunoassay (QCI) for analysis of NF‐κB DNA‐binding activity, and (b) validation of the QCI with the EMSA using nuclear and cytosolic extracts from cultured prostate cancer cells (PC3), rat liver homogenates and human lymphocytes. The QCI for analysis of NF‐κB DNA binding activity has advantages over the EMSA: (1) Higher speed: 3–5 h post sample preparation, (2) Greater sensitivity: 10 pg NF‐κB/well, (3) Quantitative: linear range: 10–1000 pg NF‐κB; r = 0.999 (4) High throughput adaptability: 96‐well plate format can analyze up to 40 samples in duplicate, (5) Safety: No radioactive isotopes, (6) Simplicity, and (7) Capability of measurement of both activated (free) NF‐κB which is translocated into the nucleus and total (bound + unbound) NF‐κB present in the cytosol/cell.
    Journal of Immunoassay & Immunochemistry - J IMMUNOASS IMMUNOCHEM. 01/2005; 26(2):125-143.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Analysis of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor produced by the cytochrome P450 pathway, presently requires high-performance liquid chromatography (HPLC) and gas chromatography/ mass spectrometry (GC/MS). To simplify 20-HETE analysis, competitive ELISAs were developed using polyclonal anti-20-HETE coated ELISA plates to which free 20-HETE and 20-HETE conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) were added. Assays were developed with and without a pro prietary enhancer solution which allows for the extraction-free measurement of 20-HETE in urine samples. The bound 20-HETE-HRP or 20-HETE-AP was detected using 3,3 ,5,5, -tetramethylbenzidine and p-nitrophenyl phosphate, respectively. Sensitivities expressed as 80% B/B0, were 0.1 ng/ml for the HRP assay, and 0 5 ng/ml for the AP assay, with r2 = 0 99 for both formats. Of the 17 lipids tested for cross-reactivity, arachidonic acid showed the highest (0.32%) followed by racemic 5-HETE (0.07%) and 8,9-dihydroxyeicosatrienoic acid (DHET) (0.04%). Preliminary validation experiments examining serum and urine concentrations of 20-HETE yield values that fall within the ranges established by GC/MS in the literature. These ELISAs provide simple and inexpensive methods for the analysis of 20-HETE in biological samples.
    Journal of Biosciences 03/2003; 28(1):109-13. · 1.76 Impact Factor
  • Matthew J. Hymes, Richard M. McGowen, Denis M. Callewaert
    [Show abstract] [Hide abstract]
    ABSTRACT: Cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of greater than 60% of prescription pharmaceuticals. Although methods involving fluorogenic substrate analogs are widely used for high-throughput screening (HTS) for CYP inhibitors, they provide less than 50% predictability compared to LC/UV. ACCURATETM, a novel fluorescent HTS assay for CYP inhibitors reactive oxygen species (ROS) during CYP3A4-mediated metabolism of testosterone, has previously been reported and shown to provide >85% predictability compared to LC/UV (Ansede, J.H., Brouwer, K.R. and Thakker, D.R. North American ISSX meeting, 2004). Given the complexity of the active site, the multiple reactions catalyzed and the multitude of CYP3A4 substrates, the ability of the ACCURATETM technology to reliably evaluate CYP3A4 metabolism and inhibition was evaluated using multiple, well-characterized substrates and inhibitors. No significant differences were observed between IC50 values obtained for using the 6β-hydroxylation of testosterone vs. the N-demethylation of dextromethorphan as the probe substrate. Comparable IC50 values were also obtained in assays using a mixture of these substrates. The largest effect on IC50 values was observed for assays conducted in the presence or absence of Cytochrome b5 with either substrate. Based on these studies, ACCURATETM provides for highly predictable HTS for CYP3A4 inhibitors using well characterized endogenous or xenobiotic substrates and eliminates the need to perform multiple assays with a range of synthetic fluorogenic substrate analogs.
    14th North American Regional International society for the study of xenobiotics Meeting;