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J D Kelly

Publications and source records attributed to J D Kelly.

At least 37 records · Page 2Linked to original sources
PubMed1999

Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent.

Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT --> TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A600-602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p < 10(-6), Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner.

Adenine↗
PubMed1998

99mTc-HL91: "hot spot" detection of ischemic myocardium in vivo by gamma camera imaging.

BACKGROUND: 99mTc-HL91 is a new hypoxia imaging agent that demonstrates increased uptake and retention in globally hypoxic myocardium in vitro. The purpose of this study was to determine whether 99mTc-HL91 could detect regional ischemia in vivo by gamma camera imaging. METHODS AND RESULTS: Eight open-chest dogs with left circumflex (LCx) stenoses were studied. Injection of 5 mCi of 99mTc-HL91 and microspheres was followed by imaging over 4 hours. Heart slices were imaged, then stained with triphenyltetrazolium chloride (TTC), and tissues were well-counted. TTC staining demonstrated no injury. Mean LCx blood flow was 0.32+/-0.04 mL x min(-1) x g(-1), and mean left anterior descending coronary artery (LAD) flow was 0.96+/-0.02 mL x min(-1) x g(-1) (ratio, 0.33). "Hot spots" were detected in 8 of 8 experiments in vivo within 60 minutes and improved over 4 hours. Region of interest analysis of LCx/LAD activity ratios demonstrated significant increases within 30 minutes (final ratio, 3.0; P<0.05). LCx and LAD washout curves demonstrated significant differences within 15 minutes. Washout curves were biexponential over 1 hour, followed by linear retention from 1 to 4 hours. Four-hour fractional retention was 0.12 for LAD and 0.44 for LCx (P<0.01). Myocardial flow versus tracer uptake demonstrated 2 phases: phase 1 (flow, 0.05 to 0.7 mL x min(-1) x g(-1)) had an inverse linear correlation (r= -0.80); phase 2, (flow, >0.7 mL x min(-1) x g(-1)) had no correlation. Ischemic heart/liver ratios remained near 1.0 for 4 hours. CONCLUSIONS: 99mTc-HL91 positively identifies regional myocardial ischemia in a canine model using 99mTc imaging. Quantitative techniques allowed identification of ischemic myocardium within 15 minutes of tracer administration.

Animals↗
PubMed1998

Signal transduction via the mitogen-activated protein kinase pathway induced by binding of coagulation factor VIIa to tissue factor.

The putative role of tissue factor (TF) as a receptor involved in signal transduction is indicated by its sequence homology to cytokine receptors (Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934-6938). Signal transduction induced by binding of FVIIa to cells expressing TF was studied with baby hamster kidney (BHK) cells stably transfected with TF and with a reporter gene construct encoding a luciferase gene under transcriptional control of tandem cassettes of signal transducer and activator of transcription (STAT) elements and one serum response element (SRE). FVIIa induced a significant luciferase response in cells expressing TF, BHK(+TF), but not in cells without TF. The BHK(+TF) cells responded to the addition of FVIIa in a dose-dependent manner, whereas no response was observed with active site-inhibited FVIIa, which also worked as an antagonist to FVIIa-induced signaling. Activation of the p44/42 MAPK pathway upon binding of FVIIa to TF was demonstrated by suppression of signaling with the specific kinase inhibitor PD98059 and demonstration of a transient p44/42 MAPK phosphorylation. No stimulation of p44/42 MAPK phosphorylation was observed with catalytically inactive FVIIa derivatives suggesting that the catalytic activity of FVIIa was obligatory for activation of the MAPK pathway. Signal transduction caused by a putative generation of FXa activity was excluded by experiments showing that FVIIa/TF-induced signaling was not quenched by tick anticoagulant protein, just as addition of FXa could not induce phosphorylation of p44/42 MAPK in BHK(+TF) cells. These results suggest a specific mechanism by which binding of FVIIa to cell surface TF independent of coagulation can modulate cellular functions and possibly play a role in angiogenesis and tumor metastasis as indicated by several recent observations.

Animals↗
PubMed1998

A chemical and genetic approach together define the biological consequences of 3-methyladenine lesions in the mammalian genome.

DNA-damaging agents produce a plethora of cellular responses that include p53 induction, cell cycle arrest, and apoptosis. It is generally assumed that it is the DNA damage produced by these agents that triggers such responses, but there is limited direct evidence to support this assumption. Here, we used DNA alkylation repair proficient and deficient isogenic mouse cell lines to demonstrate that the signal to trigger p53 induction, cell cycle arrest, and apoptosis in response to alkylating agents does emanate from DNA damage. Moreover, we established that 3-methyladenine, a relatively minor DNA lesion produced by most methylating agents (which form mainly 7-methylguanine), can specifically induce sister chromatid exchange, chromatid and chromosome gaps and breaks, S phase arrest, the accumulation of p53, and apoptosis. This study was made possible by the generation of 3-methyladenine DNA glycosylase null mutant cells by targeted homologous recombination and by the chemical synthesis of a methylating agent that almost exclusively produces 3-methyladenine DNA lesions. The combined use of these two experimental tools has defined the biological consequences of 3-methyladenine, a DNA lesion produced by endogenous cellular metabolites, environmental carcinogens, and chemotherapeutic alkylating agents.

Adenine↗
PubMed1998

Quantitative and qualitative analysis of DNA methylation at N3-adenine by N-methyl-N-nitrosourea.

The sequence-specific alkylation of DNA by N-methyl-N-nitrosourea (MNU) has been demonstrated for the minor groove N3-methyladenine (N3-MeAde) adduct using neutral thermal hydrolysis and polyacrylamide sequencing gels. The ratio of relative yields of N7- and N3-MeAde and N7-methylguanine (N7-MeGua) is approximately 0.03:0. 15:1.00, respectively, on the basis of the gel data, and these values are comparable to relative yields determined by bulk digestion of MNU-methylated DNA when HPLC was used to analyze the individual adducts. In contrast to the methylation at N7-guanine (N7-Gua) by MNU, alkylation at Ade shows minimal sequence selectivity. Similar to the methylation at N7-Gua, formation of N3-MeAde by MNU is inhibited by 50-200 mM concentrations of NaCl and DNA binding cations, including distamycin and spermine. However, N3-MeAde formation at Ade residues within methidiumpropyl-EDTA-Fe(II) footprinted distamycin DNA affinity binding regions is selectively inhibited at low concentrations of distamycin relative to Ade sites outside of ligand binding regions, and N7-Gua within or outside the distamycin binding regions. HPLC analysis shows that distamycin also quantitatively inhibits the production of N3-methylguanine when calf thymus DNA is treated with MNU or methyl methanesulfonate. The specific inhibitory effect of distamycin, which binds in the minor groove at Ade/Thy-rich sequences, provides additional evidence that the predominant DNA lesion detected at Ade by sequencing gel analysis involves minor groove N3-MeAde modifications.

Adenine↗
PubMed1998

Clinical response to an oral prostaglandin analogue in patients with interstitial cystitis.

OBJECTIVE: To assess the efficacy of the oral prostaglandin analogue misoprostol in controlling the symptoms of interstitial cystitis in patients with refractory disease. METHODS: Twenty-five patients were commenced on misoprostol 600 micrograms daily for 3 months. Patients who responded to therapy were offered treatment for a further 6 months. Assessment of the response was by a voiding log and an interstitial cystitis symptom score. RESULTS: At 3 months, 14 patients (56%) had significantly improved, and after a further 6 months, 12 patients (48%) had a sustained response. The incidence of adverse drug effects was 64%. Most side effects were minimal, and the response rate in patients who were able to tolerate the drug was 87% at 3 months and 75% at 9 months. CONCLUSIONS: The oral prostaglandin analogue misoprostol is effective in treating the symptoms of interstitial cystitis. It is possible that prostaglandins have a cytoprotective action in the urinary bladder.

Administration, Oral↗
PubMed1997

99mTc-HL91. Effects of low flow and hypoxia on a new ischemia-avid myocardial imaging agent.

BACKGROUND: 99mTc-HL91 is a potential imaging agent that has demonstrated increased uptake in hypoxic tumor cells. The purpose of this study was to determine if 99mTc-HL91 demonstrates increased uptake and retention in ischemic and hypoxic myocardium. METHODS AND RESULTS: 99mTc-HL91 (11.1 MBq) was infused over 10 minutes, followed by a 60-minute clearance phase. Activity was monitored by using an NaI detector. Three groups were studied using Krebs-Henseleit buffer (KH): controls (12 mL/min, n = 6), low-flow ischemic (1 mL/min, n = 7), and hypoxic (12 mL/min, n = 8). Two groups were perfused with KH, red blood cells, and albumin: controls (6 mL/min, n = 6) and low-flow ischemic (0.5 mL/min, n = 6). For the KH hearts, the 99mTc-HL91 peak uptake progressively increased from control (6.3 +/- 0.5 microCi, mean +/- SEM) to hypoxic (9.1 +/- 1.0 microCi) to low flow (44.0 +/- 2.6 microCi; P < .01). The peak uptake low-flow/control ratio was 7:1. Final retention increased progressively from control (0.8 +/- 0.1 microCi) to hypoxic (2.9 +/- 0.5 muCi) to low flow (10.9 +/- 1.3 microCi; P < .01). The final low-flow/control activity ratio was 13.6:1. Similar results were observed in the red blood cell-perfused control and low-flow groups. CONCLUSIONS: This study introduces a new myocardial "hot spot" imaging agent, 99mTc-HL91. This agent demonstrates increased myocardial uptake and retention in hypoxic and low-flow ischemic models. Further in vivo imaging studies are warranted to determine the clinical potential of this agent.

Animals↗
PubMed1997

Aflatoxin B1 activation in human lung.

Inhalation exposure to the carcinogen aflatoxin B1 (AFB1) in certain occupations is considerable. Because circumstantial epidemiological evidence suggests that AFB1 inhalation may cause primary lung cancer, we investigated AFB1 activation by human lung microsomes. Microsomes were incubated with [3H]AFB1 (124 microM), and activation to the AFB1-8,9-epoxide was measured as the AFB1-glutathione (AFB1-GSH) conjugate by HPLC. The formation of AFB1-GSH was in the range of 0.05-0.073 fmol/mg protein/min. The role of cytochrome P450 (CYP) 3A in this activation was investigated by oxidation of nifedipine (a prototype substrate for CYP 3A), by immunoinhibition, and by immunoblot analysis. Nifedipine oxidation varied from 0.2 to 19.2 pmol/mg protein/min in microsomes from different subjects, but did not correlate with AFB1 activation. Anti-human polyclonal CYP 3A4 IgG inhibited AFB1 activation. CYP 3A isoforms were immunoestimated to be in the range of 0.01-1.90 pmol/mg protein. Neither CYP 1A2 nor associated activity was detected in the lung microsomes. These data indicate that human lung microsomes activate AFB1 to form the exo-AFB1-8,9-epoxide and that CYP(s) of the 3A subfamily may be responsible for this activity. The relatively low amount of AFB1 activation in human lung compared to that in human liver can be explained by the scarcity of CYP-containing cells in the lung. In situ AFB1 activation and resultant carcinogenic risk are distinctly possible in occupational settings where inhalation of AFB1-contaminated dusts occurs.

Aflatoxin B1↗
PubMed1997

Validation of a rapid method to quantify apoptosis in superficial bladder cancer.

OBJECTIVES: To derive and validate a rapid method for calculating apoptotic indices in superficial transitional cell carcinoma (TCC) as a measure of chemosensitivity to mitomycin. MATERIALS AND METHODS: Apoptotic cells, identified by light microscopy in 20 superficial TCC specimens, were expressed as an index of the total tumour cell population within defined fields. For a given field, the total cell population was estimated by: (i) an exhaustive count of the total number of cells in the field and (ii) an abbreviated method in which the number of cells in a subfield was multiplied to provide an estimate of the total field number. Field and specimen estimates were compared using agreement statistics and the intra- and inter-observer reproducibility of apoptotic indices calculated. RESULTS: Cellularity and apoptotic indices obtained using method (ii) were correlated significantly with the true cell counts (P < 0.001). Agreement statistics showed that only 9.4% of counts fell outside two standard deviations (SD) from the mean in field analysis, and only 10% of counts fell outside 2 SD from the mean in specimen analysis. There was a fivefold variation in tumour cell counts among individual fields. CONCLUSIONS: The reported variation in cellularity among fields shows that the calculation of apoptosis must use the total cell population as the reference. The limits of agreement for the estimated and true cell counts are small enough to be confident that the shorter method to estimate cellularity can be used in place of counting all cells.

Antibiotics, Antineoplastic↗
PubMed1997

Symptomatic outcome following clam ileocystoplasty.

OBJECTIVE: This study was designed to assess the outcome of clam ileocystoplasty (CI) based on patients' subjective perception and objectively validated scoring systems. Quality-of-life parameters were also examined as aspects of outcome. MATERIAL AND METHODS: Between 1989 and 1994, 27 patients (15 male and 12 female) underwent CI. The median age was 41 years (13-75 years). CI was performed after failed pharmacological treatment. The mean length of follow-up for this patient cohort was 18 months (range: 1-4 years). RESULTS: Overall cure rate was objectively assessed at 61%. Seventy-two percent of patients described a subjective improvement of symptoms, and a small number of patients (12.5%) were subjectively worse following the operation. Quality of life was improved in 75% of patients, with 12.5% showing no improvement and 12.5% being worse. Fifty-six percent of patients noted a minor degree of voiding dysfunction, whereas 25% noted moderate and 19% noted severe voiding dysfunction. CONCLUSIONS: CI offered improvement with satisfaction in the group of patients studied, however not all patients achieved a satisfactory outcome. The patients who were worse following the procedure were all young females with detrusor instability. Careful patient selection and pre-operative counselling may reduce the failure rate. Outcome analysis based on standardised questionnaires allows patients and surgeons to make informed decisions with realistic expectations.

Adolescent↗
PubMed1996

Development of a SCAR marker linked to the I gene in common bean.

Two 24-mer SCAR primers (SW13) were developed from a previously identified 10-mer RAPD primer (OW13(690)) linked to the I gene, which conditions resistance to bean common mosaic virus (BCMV) in common bean. Linkage between SW13 and the I gene was tested in three F2 populations segregating for both SW13 and the I gene: N84004/Michelite (1.0 +/- 0.7 cM), Seafarer/UI-114 (1.3 +/- 0.8 cM), and G91201/Alpine (5.0 +/- 2.2 cM). SW13 proved to be more specific and reproducible than the OW13(690) RAPD marker. Using different heat-stable DNA polymerases, SW13 amplified a single 690-bp fragment linked to the I gene that more consistently permitted the identification of resistant plants. In addition, the presence of the I gene was detected using SW13 in genotypes originating from different gene pools of Phaseolus vulgaris L., indicating a broad utility of this marker for bean breeding programs.

Alleles↗