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J D Regan

Publications and source records attributed to J D Regan.

At least 37 records · Page 2Linked to original sources
PubMed1994

An acutely lethal simian immunodeficiency virus stimulates expansion of V beta 7- and V beta 14-expressing T lymphocytes.

SIVsmmPBj14, a variant simian immunodeficiency virus isolated from a pig-tailed macaque, stimulates the proliferation of macaque T lymphocytes in vitro and induces an acutely lethal disease in macaques characterized, in part, by lymphadenopathy and splenomegaly. To determine whether SIVsmmPBj14 exhibits superantigen-like activity, in vitro and in vivo studies of T-cell receptor V beta repertoire were undertaken using PCR-based quantitative methods. Whereas in vitro phytohemagglutinin stimulation of macaque peripheral blood lymphocytes did not cause a perturbation of T-cell receptor V beta repertoire, SIVsmmPBj14 stimulated the expansion of both CD4+ and CD8+ T-lymphocyte subpopulations expressing the V beta 7 and V beta 14 gene families. Such V beta 7 and V beta 14 expansions could be confirmed by a multiple RNase protection assay. Furthermore, the expansion of the same lymphocyte subpopulations was also detected in peripheral blood lymphocytes and lymph node cells of virus-infected macaques. These observations suggest that SIVsmmPBj14-mediated V beta expansion may contribute to the induction of an acutely lethal disease in macaques.

Acute Disease↗
PubMed1992

DNA as a solar dosimeter in the ocean.

Stratospheric ozone depletion may result in increased solar UV-B radiation to the ocean's upper layers and may cause deleterious effects on marine organisms. The primary UV-B damage induced in biological systems is to DNA. While physical measurements of solar UV-B penetration into the sea have been made, the effective depth and magnitude of actual DNA damage have not been determined. In the experiments reported here, UV-B-induced photoproducts (cyclobutane pyrimidine dimers) have been quantified in DNA molecules exposed to solar UV at the surface and at various depths in clear, tropical marine waters off Lee Stocking Island (23 degrees 45' N, 76 degrees 0.7' W), Exuma Cays, Bahamas. (14C)thymidine-labeled DNA or unlabeled bacteriophage phi X174 DNA was placed in specially designed quartz tubes at various depths for up to five days. Following exposure, DNA samples were removed to the laboratory where UV-B-induced pyrimidine dimers were quantified using a radiochromatographic assay, and bacteriophage DNA inactivation by solar UV-B was assayed by plaque formation in spheroplasts of Escherichia coli. Pyrimidine dimer induction was linear with time but the accumulation of dimers in DNA with time varied greatly with depth. Attenuation of dimer formation with depth of water was exponential. DNA at 3 m depth had only 17% of the pyrimidine dimers found at the surface. Bacteriophage phi X174 DNA, while reduced 96% in plaque-forming ability by a one day exposure to solar UV at the surface of the water, showed no effect on plaque formation after a similar exposure at 3 m. The data collected at the water's surface showed a "surface-enhanced dose" in that DNA damages at the real surface were greater than at the imaginary surface, which was obtained by extrapolating the data at depth to the surface. These results show the sensitivity of both the biochemical (dimers) and biological (phage plaques) DNA dosimeters. DNA dosimeters offer a sensitive, convenient and relatively inexpensive monitoring system, having both biochemical and biological endpoints for monitoring the biologically effective UV-B flux in the marine environment. Unlike physical dosimeters, DNA dosimeters do not have to be adjusted for biological effectiveness since they are sensitive only to DNA-mediated biologically effective UV-B radiation. Results of pyrimidine dimer induction in DNA by solar UV accurately predicted UV doses to the phage DNA.

Bacteriophage phi X 174↗
PubMed1990

Cyclobutane-pyrimidine dimer excision in UV-sensitive CHO mutants and the effect of the human ERCC2 repair gene.

Using a radiochromatographic assay, we have examined cis-syn cyclobutane-pyrimidine dimer removal after ultraviolet irradiation in cell lines representative of the first 6 complementation groups of Chinese hamster ovary DNA nucleotide excision repair mutants. AA8, the CHO cell line from which these mutants were derived, consistently showed normal dimer excision for a rodent cell. The mutants uniformly exhibited no significant dimer excision within the limits of determination. Additionally, V-H1, a mutant belonging to complementation group 2 and derived from V79 hamster cells, exhibited no dimer excision. Two UV5 derived transformants that carry the complementing human ERCC2 repair gene showed a capacity for dimer excision comparable to the AA8 wild-type cells.

Animals↗
PubMed1989

CHO mutant UV61 removes (6-4) photoproducts but not cyclobutane dimers.

The CHO mutant UV61 was previously assigned to complementation group 6 of UV-sensitive rodent cell mutants. UV61 is less sensitive to killing by UV radiation than mutants such as UV5, which is highly defective in the incision process that acts on UV-induced lesions. The D37 for cell survival is approximately 4 J/m2 for UV61, compared with 10 J/m2 for the parental AA8 line and approximately 2 J/m2 for UV5. Similarly, mutation induction at the hprt and aprt loci shows an intermediate response to UV61. In a post-replication recovery assay, the kinetics of maturation of pulse-labelled nascent DNA were normal after UV irradiation in UV61. Data from alkaline elution and alkaline unwinding assays showed that the rates of break accumulation and resealing, measured 0-120 min after irradiation, were also normal in the mutant. This repair incision correlated with the rapid, normal removal of pyrimidine(6-4)pyrimidone photoproducts in UV61 measured using a radioimmunoassay that is specific for this class of damage. In contrast, after exposure to 10 or 15 J/m2, no detectable removal of cyclobutane dimers from DNA was found in UV61 while AA8 cells removed 32% by 24 h. We suggest that the mutation in UV61 specifically lowers the affinity of a repair protein for cyclobutane dimers, which are also inefficiently removed from the bulk DNA of normal CHO cells. The resistance of UV61 to killing by the direct acting chemical 7-bromomethylbenz[a]anthracene was only slightly greater than that of UV5, indicating defective repair of bulky chemical adducts in addition to cyclobutane dimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Detection and repair of a UV-induced photosensitive lesion in the DNA of human cells.

Irradiation with UV light results in damage to the DNA of human cells. The most numerous lesions are pyrimidine dimers; however, other lesions are known to occur and may contribute to the overall deleterious effect of UV irradiation. We have observed evidence of a UV-induced lesion other than pyrimidine dimers in the DNA of human cells by measuring DNA strand breaks induced by irradiating with 313-nm light following UV (254-nm) irradiation. These breaks, measured by alkaline sucrose sedimentation, increased linearly with the dose of UV light over the range tested (10-40 J/m2). The breaks cannot be photolytically induced 5 h after a UV dose of 20 J/m2 in normal cells; however, in xeroderma pigmentosum variant cells, the breaks are inducible for up to 24 h after UV irradiation. Xeroderma pigmentosum group A cells in the same 5-h period show an increase in the number of strand breaks seen with 313-nm light photolysis from about 2 to 4 breaks/10(9) dalton DNA. These breaks can then be induced for up to 24 h. These data suggest that, in normal cells, the lesion responsible for this effect is rapidly repaired or altered; whereas, in xeroderma pigmentosum variant cells it seems to remain unchanged. Some change apparently occurs in the DNA of xeroderma pigmentosum group A cells which results in an increase in photolability. These data indicate a deficiency in DNA repair of xeroderma pigmentosum variant cells as well as in xeroderma pigmentosum group A cells.

DNA↗
PubMed1986

Quantitation of carcinogen-induced DNA damage and repair in human cells with the UVR ABC excision nuclease from Escherichia coli.

Recent studies by others have shown that the endonuclease complex coded for by the uvrA, uvrB and uvrC genes of Escherichia coli (UVR ABC excision nuclease) can incise DNA containing a variety of 'bulk-type' lesions, such as those resulting from u.v. light, (+/-)-7 alpha,8 beta-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), and N-acetoxy-2-acetylaminofluorene. Using partially purified UVR ABC excision nuclease, we have quantitated the number of endonuclease sensitive sites (ESS) in purified DNA isolated from human fibroblasts treated with u.v. light or BPDE. The number of ESS/10(8) daltons of DNA were calculated from the number average mol. wt. of the DNA as determined by sedimentation in alkaline sucrose gradients. The number of endonuclease sites increased linearly with increasing doses of either u.v. light or BPDE. The UVR ABC excision nuclease was able to incise a majority of the BPDE-DNA adducts. Xeroderma pigmentosum fibroblasts, complementation group A (XP12BE) had 20-25% more ESS at each dose than the BPDE-treated normal (HSBP) cells. Cells treated with 4 microM BPDE and allowed 12 h of incubation to perform excision repair showed removal of 60% of the initial number of ESS from HSBP DNA and 40% of the ESS from XP-A DNA. Beyond 12 h XP12BE cells lost no additional ESS while HSBP cells continued to lose ESS, although at a slower rate, until at 48 h only 22% of the initial ESS remained. In cells treated with 10 J/m2 of u.v. light, the UVR ABC excision nuclease detected 60% of the sites recognized by the pyridimine dimer specific Micrococcus luteus glycosylase/apyrimidinic endonuclease. These results demonstrate the potential use of the UVR ABC excision nuclease in a quantitative assay for determining the number of carcinogen-induced lesions in human DNA.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

Effects of extremely low frequency (ELF) electric fields on cell growth and DNA repair in human skin fibroblasts.

A number of physical and chemical agents in the environment have been studied for their ability to induce or alter DNA repair mechanisms in human cells. We have investigated the effects of 60 Hz, 1000 V/cm electric fields on DNA repair in normal human fibroblasts in vitro. An examination was done on the ability of electric fields suspected to cause damage which could be repaired by thymine dimer excision and measurable by the bromodeoxyuridine photolysis assay. The thymine dimer assay with enzyme-sensitive site analysis was used to measure the cells' capacity for removing ultraviolet light (u.v.)-induced pyrimidine dimers; during exposure to electric field 24 hr before u.v. irradiation; 24 hr after u.v. irradiation; and up to 48 hr continuously after u.v. irradiation. Cell growth and cell survival following electric field exposure were also studied. Within the limits of these experiments, it was found that exposure to such electric fields did not alter cell growth or survival, and no DNA repair or alteration in DNA excision repair capacity was observed as compared with unexposed control cultures.

Cell Division↗
PubMed1985

Inhibition of DNA repair by trifluoperazine.

We examined the possible role of calmodulin in the excision repair of ultraviolet light-induced pyrimidine dimers in damaged DNA by means of specialized assay systems. These assays included bromodeoxyuridine photolysis, dimer chromatography and cytosine arabinoside incorporation in conjunction with hydroxyurea. The calmodulin antagonist, trifluoperazine, and the calcium-chelating agent, EGTA, were employed to ascertain what affect calmodulin played in the repair process. Normal human fibroblast cells were used in all studies described in this report. After exposure to 10 J/m2 of 254 nm light, we observed a decrease of about 30% in the number of single-strand breaks produced in the presence of 25 microM trifluoperazine (1.9 vs. 3.3) in controls although the numbers of bases re-inserted in the repaired regions were similar (64 vs. 72). Measurement of thymine-containing dimers remaining throughout a 24 h time period indicated a 30% difference in the excision of dimers when tested with either EGTA or trifluoperazine. We also observed a significant decrease in the number of cytosine arabinoside arrested repair sites in the presence of either EGTA or trifluoperazine. The results are discussed with relation to the possibility of calmodulin altering the initial incision by repair endonuclease.

Bromodeoxyuridine↗
PubMed1985

Elevation of dCTP pools in xeroderma pigmentosum variant human fibroblasts alters the effects of DNA repair arrest by arabinofuranosyl cytosine.

DNA excision repair inhibition by arabinofuranosyl cytosine (ara-C) or by ara-C/hydroxyurea (HU) was measured in log phase and confluent cultures of normal and xeroderma pigmentosium (XP)-variant human fibroblasts following insult by ultraviolet (UV) light (20 J/m2). Repair inhibition was determined by measuring the accumulation of DNA single-strand breaks/10(8) daltons following cell culture exposure to ara-C or ara-C/HU in a series of 3 hr. pulses up ro 24 hr. after UV insult. Both normal and XP-variant derived cells showed a wide range of sensitivity to ara-C in log phase cells (0.2-9.4 breaks/10(8) daltons DNA), although strand break accumulation was constant for each specific cell line. The same cells were more sensitive to ara-C/HU with a 2-14 fold increase in DNA strand breaks depending upon the cell line assayed. In confluent cultures of normal cells, maximum sensitivity to ara-C and ara-C/HU was achieved with similar levels of repair inhibition observed (16.1 and 16.5 breaks/10(8) daltons, respectively). The same level of repair inhibition was observed in confluent XP-variants receiving ara-C/HU, but was reduced by 62-68% in cells treated with ara-C alone. Ara-C repair arrest was more rapidly reversed by competing concentrations of exogenous deoxycytidine (dCyd) in XP-variant compared to normal cells, especially in confluent cell cultures. In ara-C/HU treated cells, the level of dCyd reversal was reduced in the XP-variant when compared to cells exposed to ara-C alone. However, the same addition of HU had relatively little effect on dCyd reversal in normal cells. The measurements of dNTP levels indicate an elevated level of intracellular deoxycytosine triphosphate in XP-variant vs normal cells. The implications of these results are discussed as they relate to possible excision repair anomalies in the XP-variant.

Cells, Cultured↗
PubMed1984

The inhibition of ultraviolet radiation-induced DNA repair in human diploid fibroblasts by arabinofuranosyl nucleosides.

The antiviral compounds 9-beta-D-arabinofuranosyladenine (ara-A), 9-beta-D-arabinofuranosyl-2-fluoroadenine (FAA), 9-beta-D-arabinofuranosylhypoxanthine (ara-Hx), 9-beta-D-arabinofuranosylguanine (ara-G), 1-beta-D-arabinofuranosylthymine (ara-T), 1-beta-D-arabinofuranosyl-2'-fluorocytosine (FAC), 1-beta-D-arabinofuranosyl-2'-fluoro-5-iodocytosine (FIAC) and 1-beta-D-arabinofuranosyl-2'-fluoro-5-methyluracil (FMAU) were compared to 1-beta-D-arabinofuranosyl cytosine (ara-C) in their ability to inhibit ultraviolet (UV) light-induced DNA repair in log phase and confluent human diploid fibroblasts. Inhibition of the polymerization or ligation steps of DNA excision repair manifests itself in the form of DNA single-strand breaks which may be quantitated through velocity sedimentation analysis in alkaline sucrose gradients. In UV-irradiated quiescent, confluent human fibroblast cultures, treatment with any of the aranucleosides leads to accumulation of single-strand breaks but the effective dose for this inhibition varies greatly. The order of their effectiveness in confluent cultures was ara-C and its derivatives greater than ara-A, FAA, ara-G, ara-Hx greater than ara-T. In rapidly cycling cells on the other hand, sensitivity to repair inhibition was exhibited only in response to ara-C and FAC. If 2 mM hydroxyurea (HU) was administered with ara-A, FAA or FMAU, however, DNA strand breaks were seen. HU also increased the efficiencies of ara-C and FAC. No significant strand breaks were observed in UV-irradiated log phase cells treated with FIAC, ara-Hx, ara-G or ara-T even in the presence of HU. The efficiencies of inhibition of unscheduled DNA synthesis (UDS) and semiconservative DNA synthesis by the aranucleosides is consistent with their relative efficiencies at producing strand breaks. The ability of the aranucleosides to inhibit DNA repair is discussed with respect to their hypothesized effects on DNA metabolic processes in eucaryotic cells.

Arabinonucleosides↗
PubMed1983

A mechanism for the control of patch size in mammalian cell DNA excision repair.

The hypothesis is suggested that size of the region excised in repair of UV-induced damage in mammalian cell is determined by the occurrence at random of a recognition sequence which terminates this excision process. The statistics of first occurrence times for a specific nucleotide sequence in a random chain are derived and shown to lead to an approximately random distribution of sizes around the average. The heterogeneity in sizes arising from a model are shown not to conflict with existing measurements. A sequence of length three or four is sufficient to account for the measured average size.

Animals↗
PubMed1983

Prior exhaustive exercise and subsequent, maximal constant load exercise performance.

The effect of an elevated blood lactate concentration on maximal constant load exercise performance (MCLE) was studied in nine males. Two experimental sessions followed a continuous VO2max test, each consisting of an initial work bout at approximately 110% VO2max, a 20 min recovery, and a second maximal performance test. Blood lactate concentrations reached approximately 10 mM following the initial work bout and were altered prior to the second bout by one of two recovery patterns: 1) active recovery (AR)--pedaling at 40% VO2max or 2) passive recovery (PR)--sitting quietly. Performance time, total pedal revolutions, metabolic parameters, and heart rate (HR) were measured during each work bout. Despite significant differences in blood lactate concentration, there were no differences in work output among the performance tests (either between conditions or from pre- to post-recovery); there was a low correlation (r = 0.08) between pre-existing blood lactate concentration and work output on a subsequent maximal constant load exercise test. It was concluded that elevated blood lactate concentration does not affect maximal constant load exercise performance.

Adult↗
PubMed1982

Studies on the inhibition of repair of ultraviolet- and methyl methanesulfonate-induced damage in the DNA of human fibroblasts by novobiocin.

The antibiotic novobiocin is shown to alter the sedimentation properties of human cellular DNA in alkaline sucrose. This alteration is at least partially due to increased DNA-protein binding in the cell in the presence of novobiocin. Pyrimidine dimer analysis and repair replication studies support previous reports that novobiocin inhibits repair of UV damage in human cells but we find this block to be shortlived. It is also shown that novobiocin is ineffective at blocking "long-patch" repair induced by methyl methanesulfonate as measured both by CsCl density centrifugation and the ara-C inhibition technique. However, the accumulation of breaks in MMS-treated cellular DNA in the presence of novobiocin suggests that some "short-patch" alkylation repair may be inhibited by the antibiotic. These findings are discussed in light of the proposal that novobiocin may inhibit a DNA gyrase-like activity in human as in bacterial cells.

Cell Line↗
PubMed1982

Differential responses of log and stationary phase human fibroblasts to inhibition of DNA repair by aphidicolin.

The tetracyclic diterpenoid, aphidicolin, is an effective inhibitor of DNA repair in human cells following ultraviolet irradiation. This inhibition is very efficient in confluent resting cells but not in rapidly cycling cells as measured by (1) analysis of DNA single-strand breaks by alkaline sucrose sedimentation, (2) chromatographic analysis of pyrimidine-dimer removal, and (3) repair replication using CsCl density centrifugation. The inhibition is reversed by deoxycytidine or thymidine but not by deoxyadenosine or deoxyguanosine during the repair period. The data suggest that differences in deoxynucleoside triphosphate pools between cycling and confluent resting cells determine the different efficacies of the agent in these two situations.

Aphidicolin↗
PubMed1982

Effects of an extract from the sea squirt Ecteinascidia turbinata on DNA synthesis and excision repair in human fibroblasts.

An aqueous ethanol extract from the marine tunicate species Ecteinascidia turbinata was studied to determine its effect on semiconservative DNA synthesis in human skin fibroblast cultures as measured by [3H] thymidine uptake in acid-insoluble cell fractions. In addition, the effect of this extract on DNA excision repair in ultraviolet light (254 nm) irradiated fibroblasts was measured by the bromodeoxyuridine photolysis assay, thymine dimer chromatography, and DNA single-strand break analysis on alkaline sucrose gradients. An extract concentration of 0.5 mg/ml resulted in 91 and 98% reductions in cell growth and DNA synthesis, respectively, after 20 hr. An extract concentration of 3.0 mg/ml resulted in a 44% reduction in excision repair in ultraviolet-irradiated cells over a 6 hr repair period; this was reduced to 26% after 24 hr of repair, and to 15% after 48 hr. Repair inhibition was accompanied by an accumulation of single-strand DNA breaks which was enhanced by the addition of 2 mM hydroxyurea. These results are discussed with respect to a mechanism of action of the marine tunicate extract at the level of DNA polymerases and are contrasted with previously studied inhibitory mechanisms of arabinofuranosyl nucleosides.

Animals↗