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D J O'Kane

Publications and source records attributed to D J O'Kane.

At least 19 recordsLinked to original sources
PubMed2001

Gene expression microarrays: a 21st century tool for directed vaccine design.

DNA microarray technology is a new and powerful tool that allows the simultaneous analysis of a large number of nucleic acid hybridization experiments in a rapid and efficient fashion. The development of the DNA microarray chip has been driven by modern techniques of microelectronic fabrication, miniaturization and integration to produce what is referred to as "laboratory-on-chip" devices. The application of DNA chip technology includes the comprehensive analysis of multiple gene mutations and expressed sequences with regard to newer drug designs, host-pathogen interactions and the design of new vaccines. An advantage of microarray technology is that it can assist researchers to better define and understand the expression profile of a given genotype associated with disease, adverse effects from exposure to certain stimuli, or the ability to understand or predict immune responses to specific antigens. This paper briefly reviews DNA microarray technology and its implications with special reference to vaccine design. The technical aspects comprising array manufacturing and design, array hybridization, formatting, scanning and data handling are also briefly discussed.

Animals↗
PubMed2001

Telomerase activity in sex cord-stromal tumors of the ovary.

OBJECTIVE: Telomerase is a ribonucleoprotein that protects chromosomes from degradation and end-to-end fusions by maintaining telomere length. Studies have shown that telomerase is present in 95% of gynecologic malignancies and in 88% of epithelial ovarian carcinomas but undetectable in benign tissue. The aim of this investigation was to determine whether telomerase is present in sex cord-stromal tumors and whether telomerase activity is indicative of patient outcomes. METHODS: Forty-five consecutive sex cord-stromal ovarian tumors were analyzed by using reverse transcription-polymerase chain reaction for expression of human telomerase, human telomerase reverse transcriptase, and telomerase activity. RESULTS: Of the 29 patients with malignant cell types (granulosa cell, Sertoli-Leydig cell, and steroid cell tumors), 21 of the 28 patients (75%) available for follow-up had recurrence, with a mean follow-up of 86 months (95% CI, 36-157 months). The telomerase repeat amplification protocol assay had a sensitivity of 74% and specificity of 94% for malignancy. Patients with telomerase-positive tumors had a mean disease-free interval of 66.5 months; for those with telomerase-negative tumors the interval was 90 months. In addition, patients with telomerase-positive tumors were more likely to be dead from disease or alive with disease than those without telomerase activity, and they showed trends toward requiring a larger number of surgical procedures for the treatment of their disease. However, these trends were not statistically significant. CONCLUSION: Although activation of telomerase is clearly an important step in carcinogenesis, it is unlikely to be helpful in the clinical management of sex cord-stromal tumors of the ovary.

DNA-Binding Proteins↗
PubMed2001

A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP.

We have previously reported that Escherichia coli and mammalian cells containing a fusion protein consisting of the Renilla luciferase linked to Aequorea GFP exhibited luminescence resonance energy transfer (LRET) from luciferase to GFP in the presence of coelenterazine. In this paper, we describe the construction of two gene fusions in which the cDNA for insulin-like growth factor II (IGF-II) is connected to the cDNA for a "humanized" GFP, and the cDNA for insulin-like growth factor binding protein 6 (IGFBP-6) is linked to a cDNA encoding the Renilla luciferase (RUC). The expression of the fusion gene constructs in CHO cells resulted in single polypeptides with the molecular weights expected for IGF-II-GFP and IGFBP-6-RUC, respectively, based on the use of antibodies against GFP and Renilla luciferase. The secretion of IGF-II-GFP from CHO cells was verified by fluorescence microscopy and the presence of IGFBP-6-RUC in the culture medium was confirmed by luminometry. The interaction between the two known binding partners, IGF-II and IGFBP-6, was monitored by measuring LRET from the IGFBP-6-RUC protein to IGF-II-GFP in the presence of coelenterazine, using a low-light imaging system and spectrofluorometry. Based on these data, luciferase-to-GFP LRET holds great promise for the study of protein-protein interactions in eukaryotic cells in real time.

Animals↗
PubMed2001

Validation of a single nucleotide polymorphism genotyping assay for the human serum paraoxonase gene using electronically active customized microarrays.

OBJECTIVES: Develop a microarray based genotyping assay to detect SNPs in human paraoxonase (PON I) and compare its accuracy with DNA sequencing and RFLP based assays. DESIGN AND METHODS: Amplicons spanning the polymorphic regions of interest were genotyped by sequencing, RFLP, and microarray technology. Validation parameters included precision, linearity of signal response, and carryover between adjacent sites on the microarray. RESULTS: A 100% correlation in results obtained using DNA sequencing, RFLP and microarray technology was observed. CONCLUSIONS: The microarray technology provides an accurate and reliable assay platform with applicability in high throughput genotyping studies.

Aryldialkylphosphatase↗
PubMed2000

A comparison of cytology and fluorescence in situ hybridization for the detection of urothelial carcinoma.

PURPOSE: We determine the relative sensitivities of cytology and fluorescence in situ hybridization (FISH) for the detection of urothelial carcinoma. MATERIALS AND METHODS: A mixture of fluorescent labeled probes to the centromeres of chromosomes 3, 7 and 17, and band 9p21 (P16/CDKN2A gene) was used to assess urinary cells for chromosomal abnormalities indicative of malignancy. A total of 280 urine specimens from 265 patients, including 150 with a history of urothelial carcinoma and 115 without a history of urothelial carcinoma, were analyzed. FISH analysis was performed without prior knowledge of clinical findings, that is biopsy, cystoscopy and cytology results. A positive result was defined as 5 or more urinary cells with gains of 2 or more chromosomes. RESULTS: A total of 75 biopsies showed urothelial carcinoma at FISH analysis among the 265 patients. The sensitivity of urine cytology for pTa (36 cases), pTis (18) and pT1-pT4 (15) tumors was 47%, 78% and 60%, respectively, for an overall sensitivity of 58%. The sensitivity of FISH for pTa (37 cases), pTis (17) and pT1-pT4 (19) tumors was 65%, 100% and 95%, respectively, for an overall sensitivity of 81%. FISH was significantly more sensitive than cytology for pTis (p = 0.046), pT1-pT4 (p = 0.025), grade 3 (p = 0.003) and all tumors (p = 0.001). The specificity of cytology and FISH among patients without cystoscopic evidence of urothelial carcinoma and no history of urothelial carcinoma was 98% and 96%, respectively (p = 0.564). CONCLUSIONS: The sensitivity of FISH for the detection of urothelial carcinoma is superior to that of cytology, and the specificity of FISH and cytology for urothelial carcinoma are not significantly different. Further prospective studies are required but FISH has the potential to improve significantly the management of urothelial carcinoma.

Centromere↗
PubMed1999

Comparison of screening methods in the detection of bladder cancer.

PURPOSE: We prospectively evaluate and compare the sensitivity and specificity of urine cytology, BTA stat, NMP22, fibrin/fibrinogen degradation products (FDP), telomerase, chemiluminescent hemoglobin and hemoglobin dipstick to detect bladder cancer. MATERIALS AND METHODS: Single voided specimens were obtained from 57 patients with bladder cancer, and 139 without evidence of bladder malignancy on cystoscopy or a negative biopsy of indeterminate lesions. A cytology report was available for 125 patients and interpreted independently. BTA stat, NMP22 and FDP were analyzed according to manufacturer specifications. The telomerase assay was performed on cells collected from urine by centrifugation in preparation for polymerase chain reaction based amplification using the telomeric repeat amplification protocol assay. The chemiluminescent screening assay for hemoglobin in urine uses the pseudoperoxidase activity of hemoglobin on hydrogen peroxide and subsequent oxidation of 7-dimethylaminonaphthalene-1,2-dicarbonic acid hydrazide to generate chemiluminescence emission. Hemoglobin dipstick was interpreted as positive if the hemoglobin content in the urine was trace or greater. RESULTS: Overall sensitivity with urine cytology, BTA stat, NMP22, FDP, telomerase, chemiluminescent hemoglobin and the hemoglobin dipstick was 44, 74, 53, 52, 70, 67 and 47%, respectively. Specificity with cytology, telomerase and FDP was high (95, 99 and 91%, respectively) but BTA stat, NMP22 (optimized), chemiluminescent hemoglobin (optimized) and the hemoglobin dipstick demonstrated lower specificity of 73, 60, 63 and 84%, respectively. Stepwise logistic regression analysis revealed that for all tumors, and within each tumor grade and stage telomerase had the strongest association with bladder cancer among all tests (69% overall concordance). Telomerase was also positive in 91% of the patients (10 of 11) with carcinoma in situ. CONCLUSIONS: Urinary telomerase had the highest combination of sensitivity and specificity (70 and 99%, respectively) for bladder cancer screening in these patients. It was the strongest predictor with superior accuracy in patients with grade 1 and noninvasive tumors (pTa), and extremely useful in patients with carcinoma in situ. Telomerase appears to be promising and outperformed cytology, BTA stat, NMP22, FDP, chemiluminescent hemoglobin and hemoglobin dipstick in the prediction of bladder cancer.

Adult↗
PubMed1998

A sandwich type acridinium-linked immunosorbent assay (ALISA) detects soluble ErbB1 (sErbB1) in normal human sera.

The epidermal growth factor receptor (ErbB1) is overexpressed in various human tumor-derived cell lines and neoplasms, where it is believed that receptor dysregulation plays a role in oncogenic transformation and tumor progression. In addition to the ErbB1 holoreceptor, numerous studies demonstrate that cells synthesize soluble or secreted forms of ErbB1, i.e., sErbB1. Overexpression of ErbB1 in a variety of tumors has led us to hypothesize that sErbB levels also may be altered during oncogenesis, tumor progression, and/or metastasis; and that these molecules may be useful tumor biomarkers. To address this hypothesis we have developed an acridinium-linked immunosorbent assay (ALISA) specific for the extracellular domain of ErbB1 that can be used to quantify the levels of sErbB1 molecules in body fluids and conditioned culture media. This assay can also detect full-length ErbB1 in cell and tissue extracts. Our ALISA is characterized by high sensitivity (intra-assay LLD < 1 fmol/ml), a broad linear range (approximately 1 to 4000 fmol/ml), and good reproducibility (CVs < 10%). Specificity experiments show that this ALISA detects p170 ErbB1 and soluble forms of ErbB1 that embody extracellular subdomains I through IV, but not forms of sErbB1 lacking subdomain IV. Our ALISA does not detect full-length ErbB2, ErbB3, or ErbB4; or p105 soluble ErbB2. We report that serum sErbB1 levels of healthy women (median = 3716 fmol/ml), ranging in age from 43 to 76 years, differ significantly from those of healthy men (median = 24,512 fmol/ml), ranging in age from 25 to 79 years. Additional analyses do not indicate that serum sErbB1 levels change with age in either healthy men or women. Immunoprecipitation experiments show that monoclonal antibodies specific for extracellular epitopes of ErbB1 completely neutralize the detection of sErbB1 in normal human sera by ALISA. Finally, we show by immunoprecipitation and Western immunoblot analyses with monoclonal antibodies specific for the extracellular domain of ErbB1 that normal human female and male sera contain a approximately 110-kDa protein. We conclude that our ALISA is measuring the relative levels of this p110 sErbB1 analog in normal human sera. Our ALISA, therefore, should be useful for measuring the levels of ErbB1 and sErbB1 molecules in tumor biopsy specimens and body fluids, respectively, and for determining whether sErbB1, like ErbB1, is a useful tumor biomarker.

Acridines↗
PubMed1997

Secretion of functional Renilla reniformis luciferase by mammalian cells.

The soft coral Renilla reniformis luciferase enzyme is a monomeric soluble intracellular protein that is used increasingly as a marker of gene expression. Here the Renilla luciferase gene was engineered to encode a protein product secreted by mammalian cells. The 5' end of the Renilla luciferase gene was fused in frame with the 3' end of a short DNA sequence encoding the signal peptide from human interleukin-2 (IL-2) protein. This construct was cloned under transcriptional control of the cytomegalovirus (CMV) promoter in a mammalian expression vector. Simian COS-7 cells were transiently transfected with the construct, and light emission was measured from cell lysates and from cell culture media. The results of these experiments indicated that Renilla luciferase was secreted as a functional enzyme by mammalian cells. The advantages and disadvantages of secreted Renilla luciferase as a marker of gene expression in comparison to other secreted protein markers are discussed.

Amino Acid Sequence↗
PubMed1997

Capillary electrophoresis with laser-induced fluorescence detection for the analysis of free and immune-complexed green fluorescent protein.

Naturally fluorescing green fluorescent protein (GFP) was separated by capillary electrophoresis (CE) and detected by laser-induced fluorescence (LIF). Exploiting recombinant technology and the natural fluorescence of GFP presents the capability of preempting the need for fluorescent derivatization. Such an approach would circumvent the obstacles typically associated with covalently labeling and purifying analytes that undergo fluorescent labeling. The unique property of GFP to fluoresce naturally was combined with CE-LIF to compare GFP isoforms prepared recombinantly or in vitro with wild-type GFP isoforms isolated from native jellyfish Aequorea victoria. Second, GFP antisera were reacted with wild-type GFP and the formation of the GFP antigen-antibody complex was monitored. A simple borate buffer, pH 8.5, was ample for resolving the two isoforms of the naturally fluorescent GFP in less than 5 min. The separation of GFP from GFP-Ab was complete in less than 7 min with the individual components detectable at the picogram level. A number of factors influence CE separation and/or LIF detection including sample buffer pH and incorporation of the additive 1,4-diamino butane. Remarkably, conditions that severely impair fluorescence detection of free GFP do not diminish fluorescence detection of the GFP antigen-antibody complex in a similar manner. Thus, the antibody appears to preserve the natural fluorophore of GFP. These data lend credence to the utility of coupling naturally fluorescent GFP to the speed, automation, and reduced sample size benefits of CE-LIF analysis for efficient separation and detection of an immunoreaction. In principle, a fusion protein of antibody with GFP as the label in CE-based immunoassays offers an advantageous alternative to the fluor-labeling process usually required in LIF detection.

Animals↗
PubMed1997

A laboratorian's perspective on evaluation and implementation of new laboratory tests.

New assay development should be directed toward answering fundamental clinical questions. Caveats that must be considered before initiating assay development projects are: New assays should allow the clinician to interact with and treat a patient more effectively, thereby improving medical outcome; and new assays should facilitate recapture of system resources, enabling cost savings or reinvestment of resources. Defining the clinical questions and consideration of the caveats permit a means of prioritizing assay development activities. Laboratorians are faced with evaluating several types of development activities that lead to assay implementation in routine clinical testing. Assays can be prioritized for up-grading to newer cost-effective technologies, provided the changes maintain or improve analytical and clinical performance. Predicting which research assay will have future value is difficult when clinical performance is not fully validated. However, such assay development has the greatest potential for changing the delivery of healthcare by a clinician.

Biomarkers↗
PubMed1995

Green-fluorescent protein mutants with altered fluorescence excitation spectra.

Using random mutagenesis and visual selection of fluorescent clones, we have isolated a T203I and a E222G mutant of the Aequorea green-fluorescent protein. Each mutant has one of the two fluorescence excitation bands of the wild type deleted and retains the other without a wavelength shift. This finding is consistent with each excitation band corresponding to a distinct spectroscopic state of the chromophore. Both mutations are single amino acid exchanges which in the linear sequence are located remotely from the chromophore but in the folded protein may be situated in its vicinity. We conclude that the mutations influence the fluorescence properties by changing the interactions between the chromophore and its protein environment.

Amino Acid Sequence↗
PubMed1994

Riboflavin synthesis genes are linked with the lux operon of Photobacterium phosphoreum.

Four genes immediately downstream of luxG in the Photobacterium phosphoreum lux operon (ribEBHA) have been sequenced and shown to be involved in riboflavin synthesis. Sequence analyses and complementation of Escherichia coli riboflavin auxotrophs showed that the gene products of ribB and ribA are 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthetase and GTP cyclohydrolase II, respectively. By expression of P. phosphoreum ribE in E. coli using the bacteriophage T7 promoter-RNA polymerase system, ribE was shown to code for riboflavin synthetase, which catalyzes the conversion of lumazine to riboflavin. Increased thermal stability of RibE on expression with RibH indicated that ribH coded for lumazine synthetase. The organization of the rib genes in P. phosphoreum is quite distinct, with ribB and ribA being linked but separated by ribH, whereas in E. coli, they are unlinked and in Bacillus subtilis, RibB and RibA functions are coded by a single gene.

Amino Acid Sequence↗
PubMed1993

Crystal structure of a flavoprotein related to the subunits of bacterial luciferase.

The molecular structure of the luxF protein from the bioluminescent bacterium Photobacterium leiognathi has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 17.8% at 2.3 A resolution. The 228 amino acid polypeptide exists as a symmetrical homodimer and 33% of the monomer's solvent-accessible surface area is buried upon dimerization. The monomer displays a novel fold that contains a central seven-stranded beta-barrel. The solvent-exposed surface of the monomer is covered by seven alpha-helices, whereas the dimer interface is primarily a flat surface composed of beta-strands. The protein monomer binds two molecules of flavin mononucleotide, each of which has C6 of the flavin isoalloxazine moiety covalently attached to the C3' carbon atom of myristic acid. Both myristyl groups of these adducts are buried within the hydrophobic core of the protein. One of the cofactors contributes to interactions at the dimer interface. The luxF protein displays considerable amino acid sequence homology with both alpha- and beta-subunits of bacterial luciferase, especially the beta-subunit. Conserved amino acid residues shared between luxF and the luciferase subunits cluster predominantly in two distinct regions of the luxF protein molecule. These homologous regions in the luciferase subunits probably share a three-dimensional fold similar to that of the luxF protein.

Amino Acid Sequence↗
PubMed1992

Fluorescence study of the ligand stereospecificity for binding to lumazine protein.

6,7-Dimethyllumazine derivatives, substituted at the 8-position with aldityls or monohydroxyalkyl groups, have been examined for their binding ability to lumazine apo-protein from two strains of Photobacterium phosphoreum using fluorescence dynamics techniques. On the protein the lumazine has a nearly monoexponential decay of fluorescence with lifetime 13.8 ns (20 degrees C). In free solution the lifetime is 9.6 ns. The concentration of free and bound lumazine in an equilibrium mixture can be recovered readily by analysis of the fluorescence decay. Only the aldityl derivatives D-xylityl and 3'-deoxy-D-ribityl, having stereoconfigurations at the 2' and 4' positions identical to the natural ligand, 8-(1'-D-ribityl), show comparable dissociation constants (0.3 microM, 20 degrees C, pH 7.0). D-Erythrityl and L-arabityl have dissociation constants of 1-2 microM. All other ligands show no interaction at all or have dissociation constants in the range 6-80 microM, which can still be determined semi-quantitatively using the fluorescence decay technique. In the case of these very weakly bound ligands, unambiguous detection of bound ligand can be shown by a long correlation time (23 ns, 2 degrees C) for the fluorescence anisotropy decay. Examination of the bound D-xylityl compound's fluorescence anisotropy decay at high time resolution (< 100 ps) shows rigid association, i.e. no mobility independent of the macromolecule. All bound ligands appear to be similarly positioned in the binding site. The influence of the stereoconfiguration at the 8-position found for lumazine protein parallels that previously observed for the enzyme riboflavin synthase, where the lumazines are substrates or inhibitors. This is consistent with the finding of significant sequence similarity between these proteins. The binding rigidity may have implications for the mechanism of the enzyme.

Apoproteins↗