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At least 577 records · Page 32Linked to original sources
PubMed1999

Interactive effects of inhibitors of poly(ADP-ribose) polymerase and DNA-dependent protein kinase on cellular responses to DNA damage.

DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP) are activated by DNA strand breaks and participate in DNA repair. We investigated the interactive effects of inhibitors of these enzymes [wortmannin (WM), which inhibits DNA-PK, and 8-hydroxy-2-methylquinazolin-4-one (NU1025), a PARP inhibitor] on cell survival and DNA double-strand break (DSB) and single-strand break (SSB) rejoining in Chinese hamster ovary-K1 cells following exposure to ionizing radiation (IR) or temozolomide. WM (20 microM) or NU1025 (300 microM) potentiated the cytotoxicity of IR with dose enhancement factors at 10% survival (DEF10) values of 4.5 +/- 0.6 and 1.7 +/- 0.2, respectively. When used in combination, a DEF10 of 7.8 +/- 1.5 was obtained. WM or NU1025 potentiated the cytotoxicity of temozolomide, and an additive effect on the DEF10 value was obtained with the combined inhibitors. Using the same inhibitor concentrations, their single and combined effects on DSB and SSB levels following IR were assessed by neutral and alkaline elution. Cells exposed to IR were post-incubated for 30 min to allow repair to occur. WM or NU1025 increased net DSB levels relative to IR alone (DSB levels of 1.29 +/- 0.04 and 1.20 +/- 0.05, respectively, compared with 1.01 +/- 0.03 for IR alone) and the combination had an additive effect. WM had no effect on SSB levels, either alone or in combination with NU1025. SSB levels were increased to 1.27 +/- 0.05 with NU1025 compared with IR alone, 1.02 +/- 0.04. The dose-dependent effects of the inhibitors on DSB levels showed that they were near maximal by 20 microM WM and 300 microM NU1025. DSB repair kinetics were studied. Both inhibitors increased net DSB levels over a 3 h time period; when they were combined, net DSB levels at 3 h were identical to DSB levels immediately post-IR. The combined use of DNA repair inhibitors may have therapeutic potential.

Androstadienes↗
PubMed1999

A study of endonuclease III-sensitive sites in irradiated DNA: detection of alpha-particle-induced oxidative damage.

An important difference between chemical agents that induce oxidative damage in DNA and ionizing radiation is that radiation-induced damage is clustered locally on the DNA. Both modelling and experimental studies have predicted the importance of clustering of lesions induced by ionizing radiation and its dependence on radiation quality. With increasing linear energy transfer, it is predicted that complex lesions will be formed within 1-20 bp regions of the DNA. As well as strand breaks, these sites may contain multiple damaged bases. We have compared the yields of single strand breaks (ssb) and double strand breaks (dsb) along with those produced by treatment of irradiated DNA with the enzyme endonuclease III, which recognizes a number of oxidized pyrimidines in DNA and converts them to strand breaks. Plasmid DNA was irradiated under two different scavenging conditions to test the involvement of OH* radicals with either 60Co gamma-rays or alpha-particles from a 238Pu source. Under low scavenging conditions (10 mM Tris) gamma-irradiation induced 7.1 x 10(-7) ssb Gy/bp, which increased 3.7-fold to 2.6 x 10(-6) ssb Gy/bp with endo III treatment. In contrast the yields of dsb increased by 4.2-fold from 1.5 x 10(-8) to 6.3 x 10(-8) dsb Gy/bp. This equates to an additional 2.5% of the endo III-sensitive sites being converted to dsb on enzyme treatment. For alpha-particles this increased to 9%. Given that endo III sensitive sites may only constitute approximately 40% of the base lesions induced in DNA, this suggests that up to 6% of the ssb measured in X- and 22% in alpha-particle-irradiated DNA could have damaged bases associated with them contributing to lesion complexity.

Binding Sites↗
PubMed1997

The politics of dependency estimates: Social Security Board statistics, 1935-1939.

State theorists maintain that Social Security Board (SSB) bureaucrats managed the evolution of public welfare. Institutional politics theory more readily accepts the influences of political interest groups. SSB archival records offer an opportunity to contrast these models and explore the early measurement of old age dependency. In reports designed to protect the 1935 Social Security Act, SSB staff exaggerated the extent of dependency among elderly persons. By 1939, Board statistics clearly showed that children were more impoverished than aged persons. SSB leadership did not repudiate prior estimates and they accepted rising transfers to aged persons, largely because of the political power of interest groups interested in flat pensions. While bureaucrats attempted to control events, an institutional politics approach better explains both the pivotal role of other, political actors and the solidification of the myth of old age impoverishment.

Activities of Daily Living↗
PubMed2000

Inhibin and activin subunits are differentially expressed in endometrial cells and leukocytes during the menstrual cycle, in early pregnancy and in women using progestin-only contraception.

Inhibins and activins are dimeric hormones which share common subunits and which have diverse endocrine and paracrine roles in regulating reproductive function. Endometrial expression of inhibin alpha, ssA and ssB subunits was examined by immunohistochemistry and in-situ hybridization, across the menstrual cycle and in early pregnancy. All three subunits were found to be expressed in endometrium, primarily by glandular epithelium in the early stages of the cycle. Following the onset of decidualization, expression of alpha, ssA and ssB subunits was up-regulated in decidualized stromal cells. A marked down-regulation of alpha subunit was detected in glandular epithelium, whilst expression of ssA and ssB subunits was maintained. This pattern was consistent in decidua from early pregnancy and additionally in endometrium from women using progestin-only contraceptives, either subdermal implants (Norplant((R))) or levonorgestrel-releasing intrauterine systems (Lng-IUS). Immunostaining was also observed for both ssA and ssB subunits in subpopulations of endometrial leukocytes, identified to be distinct subsets of macrophages, neutrophils and mast cells. Potential paracrine roles for activins may be envisaged in facilitating tissue remodelling during decidualization, in tissue repair following menstruation, and additionally in modulating premenstrual inflammatory events.

Activins↗
PubMed1997

Photochemical production of uracil quantified in bromodeoxyuridine-substituted SV40 DNA by uracil DNA glycosylase and a lysyl-tyrosyl-lysine tripeptide.

Exposure to UVA radiation of SV40 DNA substituted with bromodeoxyuridine (BrdU) in the presence of Hoechst dye 33258 results in the production of uracil. The yield of uracil was determined by measuring the increase in the single-strand break (SSB) yield after incubation of the photolyzed DNA with uracil-DNA glycosylase (UDG) in the presence of the tripeptide lysyl-tyrosyl-lysine (KYK). UDG removes uracil to leave an abasic site which is then cleaved to a SSB by KYK. The SSB yield was quantified by digital video imaging of ethidium fluorescence after separation of the I, II and III forms of SV40 DNA by agarose gel electrophoresis. Uracil is not detected when photolysis is carried out in the absence of the dye nor when unsubstituted DNA is used as the substrate. Without UDG or KYK treatment, the F0 for the loss of form I DNA is 100 J/m2. This falls to 13 J/m2 after incubation with UDG and KYK, indicating that uracil formation is approximately 5-fold more efficient than SSB formation. Formation of uracil suggests a mechanism for the high cellular toxicity of the dye-BrdU-UVA treatment.

Bromodeoxyuridine↗
PubMed1985

A complex of single-strand binding protein and M13 DNA as hybridization probe.

Single-stranded DNA was complexed to the single-strand binding protein (SSB) of Escherichia coli in a mass ratio of 30:1. The protein moiety of this complex can be labelled by a number of methods of which we have chosen radio-iodination and biotinylation as examples. The SSB-M13 DNA complexes, labelled to high specific activities, were used as probes in hybridization experiments in which 1.6 X 10(-18) moles of immobilized target DNA were detected. The stability of the hybrids was not severely decreased by the binding of SSB. Analysis of hybrids by electron microscopy showed that complexing of DNA with SSB could be used to allow its subsequent identification in the hybrids.

Biotin↗
PubMed2002

Modulation of enzymatic activities of Escherichia coli DnaB helicase by single-stranded DNA-binding proteins.

The modulation of enzymatic activities of Escherichia coli DnaB helicase by homologous and heterologous single-stranded DNA-binding proteins (SSBs) and its DNA substrates were analyzed. Although DnaB helicase can unwind a variety of DNA substrates possessing different fork-like structures, the rate of DNA unwinding was significantly diminished with substrates lacking a 3' fork. A 5 nt fork appeared to be adequate to attain the maximum rate of DNA unwinding. Efficient helicase action of DnaB requires the participation of SSBs. Studies involving heterologous SSBs demonstrated that they can stimulate the helicase activity of DnaB protein under certain conditions. However, this stimulation occurs in a manner distinctly different from that observed with cognate E.coli SSB. The E.coli SSB was found to stimulate the helicase activity over a wide range of SSB concentrations and was unique in its strong inhibition of single-stranded DNA-dependent ATPase activity when uncoupled from the DNA helicase activity. In the presence of a helicase substrate, the ATPase activity of DnaB helicase remained uninhibited. Thus, E.coli SSB appears to coordinate and couple the ATPase activity to the DNA helicase activity by suppressing unproductive ATP hydrolysis by DnaB helicase.

Adenosine Triphosphatases↗
PubMed2005

The accumulation of MMS-induced single strand breaks in G1 phase is recombinogenic in DNA polymerase beta defective mammalian cells.

DNA polymerase (Pol) beta null mouse embryonic fibroblasts provide a useful cell system to investigate the effects of alterations in base excision repair (BER) on genome stability. These cells are characterized by hypersensitivity to the cytotoxic effects of methyl methanesulfonate (MMS) and by decreased repair of the MMS-induced DNA single strand breaks (SSB). Here, we show that, in the absence of Pol beta, SSB accumulate in G1 phase cells, accompanied by the formation of proliferating cell nuclear antigen foci in the nuclei. When replicating Pol beta null cells are treated with MMS, a rapid phosphorylation of histone H2AX is detected in the nuclei of S phase cells, indicating that double strand breaks (DSB) are formed in response to unrepaired SSB. This is followed by relocalization within the nuclei of Rad51 protein, which is essential for homologous recombination (HR). These findings are compatible with a model where, in mammalian cells, unrepaired SSB produced during BER are substrates for the HR pathway via DSB formation. This is an example of a coordinated effort of two different repair pathways, BER and HR, to protect mammalian cells from alkylation-induced cytotoxicity.

Animals↗
PubMed2005

Single-stranded DNA-binding protein of Deinococcus radiodurans: a biophysical characterization.

The highly conserved bacterial single-stranded DNA-binding (SSB) proteins play an important role in DNA replication, repair and recombination and are essential for the survival of the cell. They are functional as tetramers, in which four OB(oligonucleotide/oligosaccharide binding)-folds act as DNA-binding domains. The protomer of the SSB protein from the extremely radiation-resistant organism Deinococcus radiodurans (DraSSB) has twice the size of the other bacterial SSB proteins and contains two OB-folds. Using analytical ultracentrifugation, we could show that DraSSB forms globular dimers with some protrusions. These DraSSB dimers can interact with two molecules of E.coli DNA polymerase III chi subunit. In fluorescence titrations with poly(dT) DraSSB bound 47-54 nt depending on the salt concentration, and fluorescence was quenched by more than 75%. A distinct low salt binding mode as for EcoSSB was not observed for DraSSB. Nucleic acid binding affinity, rate constant and association mechanism are quite similar for EcoSSB and DraSSB. In a complementation assay in E.coli, DraSSB took over the in vivo function of EcoSSB. With DraSSB behaving almost identical to EcoSSB the question remains open as to why dimeric SSB proteins have evolved in the Thermus group of bacteria.

Amino Acid Sequence↗
PubMed1999

Antitumor activity of KF22678, a novel thioester derivative of leinamycin.

KF22678, a novel thioester derivative of leinamycin with the 1-oxo-1,2-dithiolane-3-one moiety, was examined for anti-tumor activity, toxicity in mice and activation mechanism. KF22678 showed a broad antitumor spectrum against human carcinoma xenografts (lung, colon, ovary and prostate). The efficacy of KF22678 was significantly higher than that of cisplatin. KF22678 exhibited low cross-resistance against various drug-resistant cell lines of MDR1 or MRP overexpressing human tumors, and, in addition, exhibited more potent antitumor activity in vivo than ADM against A2780/ADM and KB/MRP xenograft. DL-Buthionine sulfoximine (BSO) pretreatment significantly reduced intracellular glutathione (GSH) level in human lung carcinoma A549 cells, leading to decrease in the cytotoxicity of KF22678, whereas the cytotoxicity of melphalan was augmented by BSO pretreatment. DNA single-strand breaks (SSB) were observed in A549 cells treated with KF22678 and bleomycin. DNA SSB induced by KF22678 was greatly reduced in the presence of BSO in the cells, whereas DNA SSB induced by bleomycin was not. In addition, the antitumor activity of KF22678 against BSO-pretreated human lung carcinoma PC-9 tumor was significantly decreased. These results suggest that the activation of KF22678 by intracellular GSH might be important for DNA SSB and antitumor activity in vitro and in vivo.

Animals↗
PubMed2004

Weaving basic and social sciences into a case-based, clinically oriented medical curriculum: one school's approach.

Southern Illinois University School of Medicine recently completed its fourth year of a resource-session-enhanced, case-based, tutor-group-oriented curriculum. As an example of a curricular unit, the authors describe the implementation of the basic and clinical sciences in one of the four units in year one, and detail that unit's organization, logistics, content, rationale, and other characteristics. The Sensorimotor Systems and Behavior (SSB) unit is preceded by a cardio-respiratory-renal unit and is followed by an endocrine-reproductive-gastrointestinal unit. A Doctoring unit temporally spans each of these three units. The SSB unit is allotted an 11.5-week period that includes an aggregate of 2.5 weeks of available clinical time, 1.5 weeks for examinations and exam study time, and approximately 8.5 weeks for tutor-group sessions, mandatory laboratory sessions, and self-directed learning. Optional resource sessions are offered during a two- to four-hour block on a single morning each week. Clinical training in the SSB unit augments self-directed, laboratory, and tutor-group learning of neuroscience, gross anatomy, cell biology, physiology, biochemistry, behavioral and social science, embryology, limited pharmacology and genetics, and basic clinical neurology for first-year students. Although it is fast-paced and places heavy responsibility for independent learning on the students, the SSB unit culminates in significant achievement in the basic and clinical sciences. The unit provides substantial clinical training and practical experience in physical and neurological examinations that directly integrate with basic science knowledge. The unit reduces lecture-based instruction, demands self-determination, and promotes experience in team effort, professionalism, peer interaction, empathy in clinical medicine, and practical use of basic science knowledge.

Curriculum↗
PubMed1974

Direct enzymatic repair of deoxyribonucleic acid single-strand breaks in dormant spores.

With the alkaline sucrose gradient centrifugation method, it was found that dormant spores of Clostridium botulinum subjected to 300 krads of gamma radiation showed a distinct decrease in deoxyribonucleic acid (DNA) fragment size, indicating induction of single-strand breaks (SSB). A two- to threefold difference in radiation resistance of spores of two strains of C. botulinum, 33A (37% survival dose [D(37)] = 110 krads) and 51B (D(37) = 47 krads), was accompanied by relatively larger DNA fragments (molecular weight 7.9 x 10(7)) obtained during extraction from the radiation-resistant strain 33A and smaller DNA fragments (molecular weight 1.8 x 10(7)) obtained under identical conditions from radiation-sensitive strain 51B. The apparent number of DNA SSB produced by 300 krads in strains 33A and 51B was 0.37 and 3.50, respectively, per 10(8) daltons of DNA. Addition of 0.02 M ethylenediaminetetraacetic acid (EDTA) to spore suspensions during irradiation doubled the apparent number of SSB in strain 33A but had no effect on strain 51B. In vivo, 0.02 M EDTA present during irradiation to 100 to 300 krads decreased survival of spores of 33A by about 30% but had little or no effect on 51B. Survival of 33A was also reduced by about 45% when the spores were irradiated while frozen in dry ice (-75 C) and, after irradiation, immediately exposed to 0.03 M EDTA for 1 h to inhibit repair in the dormant spores. These results suggest that the highly radiation-resistant strain 33A may be able to accomplish repair of SSB during irradiation or after irradiation under nonphysiological conditions, i.e., in the dormant state. This repair can be inhibited by EDTA. Sedimentation patterns show that DNA from spores of both strains 33A and 51B did not show any postirradiation repair during the first 6 h of germination, as opposed to Bacillus subtilis spores, which exhibit repair immediately after germination. These observations suggest the existence of direct repair in physiological dormant spores of strain 33A in the cryptobiotic resting state in the absence of germination. The repair seems to be similar to that of polynucleotide ligase activity shown to be operative in some vegetative cells. Apparently radiation-sensitive strains such as 51B and B. subtilis are generally poor in DNA repair enzyme activity under conditions of spore dormancy, which may account for the approximately threefold difference in radiation sensitivity or DNA fragility of different strains, or both.

Cell Survival↗
PubMed1995

Preliminary in vitro investigation into the use of alkaline elution assay for the biomonitoring of humans exposed to genotoxic agents.

1. This in vitro study was undertaken as a preliminary approach before assessing whether the alkaline elution assay can be applied to peripheral blood lymphocytes (PBL) for the monitoring of humans exposed to genotoxic agents such as polycyclic aromatic hydrocarbons (PAH). We have compared in vitro, with the aid of the alkaline elution assay, the formation and the repair of DNA single-strand breaks (ssb) induced by different genotoxic agents [gamma-irradiation, ethyl methanesulfonate (EMS), benzo pyrene diol epoxide (BPDE)] on quiescent and PHA-stimulated human lymphocytes and on a fibroblast cell line. 2. Gamma-irradiation (4 Gy) induced an equivalent amount of DNA ssb in the three cell types. On the other hand, after treatment with EMS (10 mM) and BPDE (50 microM), a higher production of DNA ssb was observed in replicating cells (PHA-stimulated lymphocytes and fibroblasts) when compared with quiescent lymphocytes. 3. After gamma-irradiation, all cell types repaired more than 65% of ssb within 1 h. After treatment with EMS, we noted a deficient DNA repair capacity in quiescent lymphocytes in comparison with replicating cells. In all cell types treated with BPDE, more breaks were observed after a 2 h repair period than immediately after treatment, demonstrating the involvement of a slow repair mechanism after BPDE treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2004

Pain relief after arthroscopic shoulder surgery: a comparison of intraarticular analgesia, suprascapular nerve block, and interscalene brachial plexus block.

In this prospective, randomized, blinded study, we assessed the analgesic efficacy of interscalene brachial plexus block (ISB), suprascapular nerve block (SSB), and intraarticular local anesthetic (IA) after arthroscopic acromioplasty. One-hundred-twenty patients were divided into 4 groups of 30. In Group SSB, the block was performed with 10 mL of 0.25% bupivacaine. In Group IA, 20 mL of 0.25% bupivacaine was administered intraarticularly at the end of surgery. In Group ISB, the block was performed with 20 mL of 0.25% bupivacaine. A control group was included for comparison. General anesthesia was administered to all patients. Patients were observed during the first 24 h. Pain scores, supplemental analgesia, satisfaction scores, and side effects were recorded at 4 and 24 h. No significant difference was observed between the IA and control groups. When compared with these groups, Groups SSB and ISB had significantly lower pain scores. At 4-h follow-up, better pain relief on movement was noted in Group ISB than in Group SSB. When compared with controls, a significant reduction in morphine consumption and a better satisfaction score were noted only in Group ISB. We conclude that ISB is the most efficient analgesic technique after arthroscopic acromioplasty. SSN block would be a clinically appropriate alternative.

Adult↗
PubMed1996

Dimethylarsenics reveal DNA damage induced by superoxide anion radicals.

We previously reported that DNA single-strand breaks (ssb) induced by exposure to dimethylarsinic acid (DMAA) were enhanced by the presence of paraquat (PQ), a superoxide anion radical 0 -(2)-producing agent, in cultured human alveolar type II (L-132) cells in vitro. In the present study, we examined the effect of sequential exposure of the cells to PQ and then DMAA under conditions causing no ssb by each alone, and observed a remarkable occurrence of ssb. The result suggests that 0 -(2) caused DNA damage, which became detectable as ssb by the treatment using DMAA. The DNA damage induced by the exposure to PQ alone was different from that by DMAA; PQ-induced damage was fully repaired after 24 h, while DMAA-induced damage was repaired only partially, and aphidicolin, an inhibitor of repair-serving DNA polymerases alpha, delta and/or epsilon, inhibited only the latter repair but not the former. These findings indicate that the DMAA treatment may be an effective tool to reveal DNA damage induced by 0 -(2), which to date has not been sufficiently clarified.

Aphidicolin↗
PubMed1997

Breaks in double-strand DNA by Cu(II) complexes of etoposide (VP-16) and its derivatives, as evaluated by S1 nuclease treatment.

Single-strand breaks (ssb) in double-strand (ds) DNA produced by hydroxyl radicals (.OH) generated by Cu(II) complexes of podophyllotoxin (PD)-related compounds were evaluated using S1 nuclease digestion. Cu(II) complexes of VP-16 (etoposide, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside)), 4'-demethylepi-PD (DEPD), and syringic acid (SA) exhibited both ssb and ds breaks (dsb) in ColE1-HaeII and pBR322-BglI DNA fragments, in which the number of ssb was found to be more than three times and four times greater than that of dsb, respectively. Cytosine (C)-methylation of cytosine-guanine doublet (CpG) in pBR322-BglI DNA inhibited both ssb and dsb within DNA segments by .OH generated by the Cu(II) complexes.

Antineoplastic Agents, Phytogenic↗
PubMed2003

Baicalin promoted the repair of DNA single strand breakage caused by H2O2 in cultured NIH3T3 fibroblasts.

The protective effect of Baicalin, a flavonoid isolated from the root of Scutellaria baicalensis G., on H2O2-induced DNA single strand break (SSB) was examined in NIH3T3 mouse fibroblasts by Comet assay (single cell gel electrophoresis technique). When the cells were pulse-chased with H2O2 (0.1-0.5 mM) for 15 min in fetal bovine serum (FBS)-free Dulbecco's Modified Eagle's Medium (DMEM), SSB occurred in the DNA as reported elsewhere in dose-dependent manner. Baicalin (50, 100 micro M) which was incubated with the cells for 24 h before the H2O2 chase did not give rise to significant protection against the SSB formation. However, when the time required to cause a change in the DNA damage histogram obtained by the Comet assay was precisely examined after the H2O2 chase, it was found that the H2O2 induced SSB was more promptly repaired in the cells pretreated with Baicalin prior to the H2O2 chase, compared to untreated control cells. At the same time, the cell viability examined by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) (MTT) assay after the H2O2 abuse was moderately recovered in the Baicalin increased by the Baicalin treatment. It was thus concluded that Baicalin that was known as an antioxidant flavonoid in vitro also functions as a biological response modifier, improving the cellular repair potential of oxidatively damaged DNA.

3T3 Cells↗
PubMed1994

Single- and double-strand breaks in pBR322 DNA by vacuum-UV from 8.3 to 20.7 eV.

Solid pBR322 DNA was irradiated in a vacuum by monochromatic photons from 8.3 eV (150 nm) to 20.7 eV (60 nm), and the formation of single- and double-strand breaks (ssb and dsb) was determined using agarose gel electrophoresis. The action cross sections increased 20 times (ssb) and 43 times (dsb) from 8.3 eV to 20.7 eV; the quantum yields increased 5.4 times (ssb) and 12 times (dsb). The cross sections for dsb were 0.0059 at 8.3 eV and 0.013 at 20.7 eV of those of ssb; these values were smaller than about 0.04 for 2.1 keV photons, or about 0.18 for 60Co gamma-rays. Although vacuum UV photons were proved to induce dsb, they had a lower efficiency than when using soft X-rays or gamma-rays.

DNA↗