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At least 793 records · Page 44Linked to original sources
PubMed1989

Measurement of DNA damage and cell killing in Chinese hamster V79 cells irradiated with aluminum characteristic ultrasoft X rays.

Chinese hamster V79 cells were irradiated with 1.487 keV aluminum characteristic X rays produced using a cold-cathode discharge tube. Under aerobic conditions a relative biological effectiveness (RBE) of 2.18 for cell killing in comparison to 250-kVp X rays was measured using cells grown in suspension and irradiated on membrane filters. DNA damage in the form of single-strand (ssb) and double-strand breaks (dsb) was measured using the filter elution technique. The aerobic RBEs are 1.64 for dsb induction and 0.49 for ssb induction, consistent with the view that dsb are more closely related to cell kill than ssb. A reduced oxygen enhancement ratio (OER) for cell killing was measured for Al-K X rays, but the OER for dsb induction was similar to that measured for 250-kVp X rays. A curvilinear relationship between dsb induction and dose is observed, similar to that seen for 250-kVp X rays. This agrees with the concept that ultrasoft X rays produce critical lesions similar to hard X rays but with a greater efficiency per unit dose.

Animals↗
PubMed1987

In vitro toxicity and DNA cleaving capacity of benzisoquinolinedione (nafidimide; NSC 308847) in human leukemia.

Benzisoquinolinedione (nafidimide; NSC 308847) is an investigational drug currently in phase I clinical testing. We have studied the antileukemic activity in vitro, the cellular drug transport, and the molecular mechanism of action with DNA of this new compound. By agarose gel electrophoresis, we verified that nafidimide is an intercalating agent, through its alteration of the electrophoretic migration of DNA products produced by the relaxing action of DNA topoisomerase I. Concentrations of up to 100 microM of nafidimide did not produce topoisomerase I-mediated DNA cleavage. Nafidimide produced DNA single-strand breaks (SSB), double-strand breaks, and DNA-protein cross-links in human myeloid leukemia cells (measured with filter elution). The ratio of SSB/DNA-protein cross-links was 1.32 +/- 0.36, a value similar to that produced by 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), suggesting that nafidimide, like m-AMSA, produced protein-associated DNA-strand breaks through a topoisomerase II-mediated reaction. The production of double-strand breaks by nafidimide also suggests the involvement of topoisomerase II in the drug-induced DNA cleavage. The cytotoxic activity of nafidimide was quantified in human myeloid leukemia cell lines differing by a factor of 70 in their cytotoxic sensitivity to m-AMSA. The m-AMSA-resistant line was less than 2-fold resistant to nafidimide. Cellular drug uptake was rapid and reached a steady state level in 30 min at 37 degrees C. At the end of exposure, drug egress was rapid, as was the disappearance of the DNA SSB. Rapid cellular uptake of nafidimide, with low retention at the end of exposure and rapid rejoining of DNA SSB suggest that prolonged cellular exposure may be necessary for optimal antitumor effect. In vitro cloning data suggest that nafidimide may be a therapeutic option for patients with leukemia resistant to m-AMSA.

Adenine↗
PubMed1988

Induction and rejoining of gamma-ray-induced DNA single- and double-strand breaks in Chinese hamster AA8 cells and in two radiosensitive clones.

The induction and rejoining of gamma-ray-induced DNA strand breaks were measured in a Chinese hamster ovary cell line, AA8, and in two radiosensitive clones (EM9 and NM2) derived from it. The kinetics of recovery from sublethal damage (SLD) and potentially lethal damage (PLD) has previously been characterized in each of these lines [vanAnkeren et al., Radiat. Res., 115, 223-237 (1988)]. No significant differences were observed among the cell lines in the yields of either DNA single-strand breaks (SSBs) or double-strand breaks (DSBs) as assayed by filter elution. Data for SSB rejoining in AA8 and NM2 cells irradiated with 7.5 Gy were fit by a biexponential process (t1/2 values of approximately 4 and 80 min). In comparison, SSB rejoining in EM9 cells was initially slower (t1/2 = 10 min) and a higher level of SSBs was unrejoined 6 h after irradiation. DSB rejoining in AA8 cells assayed at pH 9.6 was also biphasic (t1/2 values of 15 and 93 min), although when assayed at pH 7.0, most (approximately 80%) of the damage was rejoined at a constant rate (t1/2 = 45 min) during the first 2 h. EM9 cells exhibited a slower initial rate of DSB rejoining when assayed at pH 9.6 but showed no difference compared with AA8 cells in DSB rejoining when assayed at pH 7.0. These results indicate that radiosensitive EM9 cells, whose kinetics of recovery from SLD and PLD was the same as that of AA8 cells, have a defect in the fast phase of SSB rejoining but no measurable defect in DSB rejoining. Conversely, NM2 cells, which displayed a reduced shoulder width on their survival curve and decreased recovery from SLD, had no demonstrable defects in the rate or extent of rejoining of DSBs or SSBs. When compared with the SLD and PLD data reported previously, these results suggest that there is no direct correlation between either of these recovery processes and the rejoining of SSBs or DSBs as assayed here.

Animals↗
PubMed1988

In vivo DNA damage and resistance to 1-methyl-1-nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea in L1210 leukemia cells.

Damages in secondary DNA structure and inactivation or activation of some repair enzymes such as DNA polymerases alpha and beta and poly(ADP-riboso)polymerase induced by 1-methyl-1-nitrosourea (MNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) after administration of the drugs at therapeutic single doses to mice bearing parent L1210 leukemia cells (L1210/0) and MNU- and BCNU-resistant L1210 leukemia cells (L1210/MNU and L1210/BCNU) were studied. Damages in DNA structure of all three leukemia strains were investigated using centrifugation on alkaline or neutral sucrose gradients. More MNU-induced single-strand breaks (SSB) and alkali-labile lesions in L1210/0 and L1210/MNU cells were revealed in newly replicated DNA as compared with those in preexisting DNA. BCNU induced fewer SSB in newly replicated DNA of L1210/0 cells than MNU. The fastest repair of the damages in newly replicated DNA was detected in L1210/BCNU and especially in L1210/MNU cells as compared with L1210/0 cells. These results suggest that there are prone errors in the repair of DNA template as many SSB were revealed in the newly replicated DNA synthesized on the repaired DNA. Repair of DNA damages in L1210/BCNU and especially in L1210/MNU cells was accompanied by activation of DNA polymerases alpha and beta and poly(ADP-riboso)polymerase. It was shown that both DNA polymerases alpha and DNA polymerase beta were involved in the repair of damages induced by MNU and only DNA polymerase beta was involved in repair of damages induced by BCNU.

Animals↗
PubMed1988

Single-strand breaks in DNA of peripheral lymphocytes of styrene-exposed workers.

Single-strand breaks (SSB) were determined by means of the DNA unwinding technique in peripheral lymphocytes of styrene-exposed workers and referents. A slight increase in SSB was observed among exposed subjects in comparison with a control group of office workers. A correlation was found between SSB, the excretion of mandelic acid and the concentration of styrene glycol in blood. The present work suggests that the DNA unwinding technique can be used for screening workers exposed to genotoxic compounds.

DNA Damage↗
PubMed1988

Induction of single-strand breaks in liver DNA of mice after inhalation of vinyl chloride.

NMRI female mice were exposed to 100, 250 and 500 ppm vinyl chloride (VC). Cell nuclei were prepared from the liver, and single-strand breaks (SSB) were determined by the DNA unwinding technique. Haemoglobin (Hb) was isolated from the blood, and the degree of alkylation was determined as a measure of in-vivo dose by means of a gas chromatography-mass spectrometry (GC-MS) technique. A maximum level of SSB in liver DNA and of adduct levels of Hb was reached at 500 ppm, indicating that saturation of metabolic activation of VC had been achieved. The results demonstrate that VC induces SSB in liver DNA of mice in a dose-dependent manner and that about 80% of the damage is repaired within 20 h.

Administration, Inhalation↗
PubMed1987

Investigation of the role of individual tryptophan residues in the binding of Escherichia coli single-stranded DNA binding protein to single-stranded polynucleotides. A study by optical detection of magnetic resonance and site-selected mutagenesis.

Fluorescence and optical detection of triplet state magnetic resonance (ODMR) spectroscopy have been employed to study the complexes formed between single-stranded polynucleotides and Escherichia coli ssb gene products (SSB) in which tryptophans 40, 54, and 88 are selectively, one residue at a time, replaced by phenylalanine using site-specific oligonucleotide mutagenesis. Fluorescence titrations and ODMR results indicate that tryptophans 40 and 54 are the only tryptophan residues in E. coli single-stranded DNA binding protein that are involved in stabilizing the protein-nucleic acid complexes via stacking interactions. Wavelength-selected ODMR measurements on E. coli SSB reveal the presence of two spectrally distinct tryptophan sites (Khamis, M. I., Casas-Finet, J. R., and Maki, A. H. (1987) J. Biol. Chem. 262, 1725-1733). Our present results indicate that tryptophan 54 belongs to the blue-shifted site, while tryptophan 40 belongs to the red-shifted site of the protein.

Amino Acid Sequence↗
PubMed1987

Comparison of induction of DNA single-strand breaks and initiation of rat hepatocarcinogenesis by different nitrosamines.

The capacities of nitrosamines to induce DNA single-strand breaks (SSB) and to initiate carcinogenesis in rat liver were compared. N-Nitrosodiethanolamine (NDELA), N-nitrosoethylhydroxyethylamine (NEHEA) and N-nitrosodiethylamine (NDEA) were equipotent in inducing DNA SSB when administered by gavage at doses of 0.35 mmol/kg, 0.015 mmol/kg and 0.37 mmol/kg, respectively. Male Wistar rats were injected with these nitrosamines and were then submitted to a selection procedure. Ten rats per group were sacrificed one week after the end of the selection to see the effects of the nitrosamines on the development of preneoplastic lesions. The numbers of gamma-glutamyl transferase (GGT)-positive lesions per cm2 were 0.8, 2.1, 5.2 and 40.1 in rats treated with saline, NDELA, NEHEA and NDEA, respectively. N-Nitrosobis(2,2,2-trifluorethyl)amine (6F-NDEA), a nongenotoxic and noncarcinogenic nitrosamine, induced 0.7 GGT-positive lesions per cm2. Ten rats per group also received 0.05% phenobarbital in their drinking-water and were killed six months after initiation in order to see the effect of the different nitrosamines on the incidence and yield of tumours. Two extrahepatic cancers were found after administration of NEHEA, whereas two hepatocellular carcinomas were detected after injection of NDEA. No cancer developed in the other groups. Although other factors may influence the process, these results indicate that no simple correlation can be established between induction of SSB in DNA and initiation of tumours by nitrosamines in rat liver.

Animals↗
PubMed1984

The contribution of hydroxyl radical to radiosensitization: a study of DNA damage.

Using the radioprotector dimethylsulfoxide, DMSO, as a scavenger of hydroxyl radicals, the proportions of DNA damage caused by OH. were determined in mammalian cells irradiated in hypoxia with or without the radiosensitizers misonidazole and TAN or in air. Yields of both single-strand breaks (SSB) and base/sugar damage (MLS for Micrococcus luteus sensitive sites) were measured for each situation. Most of the damage enhanced by the sensitizers was found to be OH. dependent, for both MLS and SSB classes of damage: most breaks (greater than 80%) enhanced by oxygen and about two-thirds of the breaks enhanced by misonidazole (hypoxia) occur at OH.-damaged sites; most if not all base/sugar damage enhanced by the sensitizers misonidazole and TAN (in hypoxia) occurs only in the presence of OH., whereas in air, some (about one-quarter) of the enhanced MLS damage does not require OH.. The sensitizer enhancement ratios in the presence of scavenger and the degree of protection afforded by the scavenger determined for total (MLS + SSB) damage agree well with those derived from corresponding survival experiments.

Animals↗
PubMed1980

A prepriming DNA replication enzyme of Escherichia coli. II. Actions of protein n': a sequence-specific, DNA-dependent ATPase.

Protein n' of Escherichia coli is required for formation of the prepriming complex in replication of the single-stranded circle of phiX174 DNA. The protein, purified to near homogeneity, possesses ATPase (dATPase) activity in the presence of single-stranded, but not duplex, DNAs. Except for phiX174 DNA, ATPase activity is completely suppressed by coating the DNA with single strand binding protein (SSB). phiX174 DNA possesses a unique sequence with a potential hairpin structure that is recognized as an effector (Shlomai, J., and Kornberg, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 799-803). Sequences with secondary structure in SSB-coated M13 DNA which are recognized by RNA polymerase, and in coated G4 DNA by primase, are inert for protein n'. Approximately 30 of the 180 molecules of SSB bound to phiX DNA are destabilized by protein n' in an ATP-dependent reaction. These actions by protein n' may be important in recognizing an origin for forming the prepriming complex that leads to initiation of phiX complementary strand synthesis.

Adenosine Triphosphatases↗
PubMed1980

A temperature-sensitive single-stranded DNA-binding protein from Escherichia coli.

A temperature-sensitive single-stranded DNA-binding protein (SSB) has been purified from mutant Escherichia coli (ssb-1) cells by use of affinity chromatography on blue dextran-Sepharose. An altered amino acid sequence in the mutant protein is apparent in tryptic digests, confirming that the ssb mutation is in the structural gene. The mutant protein is less effective than the wild type in protecting single-stranded DNA from nuclease S1 digestion and in inhibiting DNA-dependent ATPases. The purified protein supports replication of phage G4 DNA in vitro at 30 degrees C, although higher levels of mutant protein, 4-fold higher than wild type, are needed to do so. The mutant protein becomes less active in supporting replication above 30 degrees C and becomes inactive at 42 degrees C within 1 min. Activity is restored upon return to 20 degrees C. Despite its temperature sensitivity in vivo and in vitro, the mutant binding protein can renature fully after exposure to 100 degrees C. Thus, the mutant protein is both heat-stable and functionally temperature-sensitive.

DNA Helicases↗
PubMed1981

Mechanism of dnaB protein action. IV. General priming of DNA replication by dnaB protein and primase compared with RNA polymerase.

The general priming system of dnaB protein and primase (Arai, K., and Kornberg, A. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4308-4312) when compared with priming by RNA polymerase shows a number of striking differences. The general priming system is initiated primarily at single-stranded region(S), being active only on single-stranded DNAs (phages and homopolymers) and inhibited by single-stranded DNA binding protein (SSB). Transcripts are only 10 to 60 residues long. By contrast, RNA priming by RNA polymerase is initiated at base-paired regions that are not destabilized by SSB (Geider, K., Beck, E., and Schaller, H. (1978) Proc. Natl. Acad. Sci. U. S. A. 76, 645-649) and transcripts on DNA not coated with SSB are generally longer. In general priming, ATP (or GTP) has three functions: (i) an allosteric effect on dnaB protein in which the nonhydrolyzed analogs adenosine-5'-O-(3'-thiotriphosphate) (or guanosine-5'-O-(3'-thiotriphosphate) can substitute, (ii) initiation of primer synthesis which can incorporate deoxy-, as well as ribonucleotides, and (iii) elongation of the primer, in which the beta, gamma-imido analog can replace ATP (or GTP). An allosteric effect of ATP on RNA polymerase has not been demonstrated, nor has the facile synthesis of hybrid transcripts of ribo- and deoxyribonucleotides.

Bacterial Proteins↗
PubMed1983

ADP-mediated dissociation of stable complexes of recA protein and single-stranded DNA.

The complete exchange of strands between circular single-stranded and full length linear duplex DNAs promoted by the recA protein of Escherichia coli is dependent upon the hydrolysis of ATP and is strongly stimulated by the single-stranded DNA binding protein (SSB). In the presence of SSB, stable complexes of recA protein and single-stranded DNA are formed as an early step in the reaction. These complexes dissociate when the ADP/ATP ratio approaches a value of 0.6-1.5, depending upon reaction conditions. Thus, ATP hydrolysis never proceeds to completion but stops when 40-60% of the input ATP has undergone hydrolysis. recA protein can participate in a second round of strand exchange upon regeneration of the ATP. While 100-200 mol of ATP are hydrolyzed/mol of heteroduplex base pair formed under standard reaction conditions in the presence of SSB, this value is reduced to 16 at levels of ADP lower than that required to dissociate the complexes. ATP hydrolysis appears to be completely irreversible since efforts to detect exchange reactions using 18O probes have been unsuccessful.

Adenosine Diphosphate↗
PubMed1984

Cervical vertebral interbody fusion in the horse: a comparative study of bovine xenografts and autografts supported by stainless steel baskets.

A modified form of the Cloward technique for anterior cervical fusion in human beings was used in the application of different grafts for evaluation of their effectiveness in stabilizing equine cervical vertebrae. Results of bovine xenograft implants in 8 horses were compared with results of stainless steel baskets (SSB) packed with cancellous autogenous bone in 8 horses. Graft material was incorporated in all cases. Evidence of graft rejection was not present. Both forms of implants decreased the mobility of the intervertebral space in which they were implanted. A fibrous connective tissue union was prevalent after xenograft implantation. Osseous union was prevalent after implantation of the SSB. Reduction in range of motion was greatest at those sites with the SSB.

Animals↗
PubMed1978

DNA strand breakage and repair in human kidney cells after exposure to incorporated iodine-125 and cobalt-60 gamma-rays.

Biological effects of 125I incorporated into DNA exceed those to be expected from the absorbed radiation dose by a factor 3--30. The reason for this discrepancy suggests special mechanisms introduced by 125I transmutation, decays by K-capture leading to the emission of an average of 6 low energy electrons including Auger electrons and to a highly positively charged daughter nuclide. The effect of 125I decay on DNA strand breakage and subsequent repair was studied. Human kidney cells T in the deep frozen state (-196 degrees C) were exposed to incorporated 125I and 60Co gamma-rays. The number of DNA single strand breaks (ssb) was determined by DNA centrifugation in alkaline sucrose gradients. DNA repair was studied by incubating the cells after thawing at 37 degrees C. For 125I decay in frozen cells which were kept with or without dimethyl-sulfoxide, 4 and 6 ssb were measured per decay. The gamma-rays produced 1ssb per 26 eV absorbed energy. Most of this damage was repaired 30 to 40 min after onset of incubation. No repair of the damage caused by 125I was observed. The high efficiency of 125I decays for the production of unreparable ssb provides evidence for the high radiotoxicity of this isotope. The observed lack of repair may in part be due to the high numbers of at least 2,000 125I decays per cell nucleus necessary for the assay system. Damage from many 125I decays may interfere with enzymatic repair processes.

Cell Nucleus↗
PubMed1984

Glutathione-dependent yield and repair of single-strand DNA breaks in irradiated cells.

The yield and rejoining of single-strand DNA breaks (ssb) was investigated after irradiation of cells which were deficient in glutathione (GSH) either due to a genetic defect of their GSH synthetase activity, or inhibition of gamma-glutamylcysteine synthetase activity by DL-buthionine-SR-sulfoximine (BSO). The results were concordant in indicating that decreased cellular GSH content is associated with an increased yield of ssb after anoxic, but not after aerobic radiation exposures. Rejoining of ssb was delayed and incomplete during a one hour's incubation period after oxic, but not after anoxic exposure of GSH-deficient cells. The defective rejoining capacity of these cells was restituted to nearly normal by the admixture of GSH-proficient cells in the incubation medium.

Buthionine Sulfoximine↗
PubMed1984

Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides. Identification of phenylalanine 60 as the site of cross-linking.

The single-stranded DNA-binding proteins from bacteriophage T4, F plasmid, Escherichia coli, and calf thymus can all be covalently cross-linked in vitro to thymine oligonucleotides by irradiating the respective protein-oligonucleotide complexes with ultraviolet light. More extensive studies on the E. coli single-stranded DNA-binding protein (SSB) indicate that this reaction is dependent upon both the length of the oligonucleotide and the dose of ultraviolet irradiation. Using anion-exchange and reverse-phase ion-pairing high-performance liquid chromatography we have isolated a specific cross-linked tryptic peptide comprising residues 57-62 of the SSB protein with the sequence valine-valine-leucine-phenylalanine-glycine-lysine. Solid-phase sequence analysis of the covalent [32P] p(dT)8-peptide complex indicates that phenylalanine 60 is the site of cross-linking. This amino acid is located within the general region of SSB (residues 1-115) that has previously been shown to contain the DNA-binding site (Williams, K. R., Spicer, E. K., LoPresti, M. B., Guggenheimer, R. A., and Chase, J. W. (1983) J. Biol. Chem. 258, 3346-3355). The high-performance liquid chromatography purification procedure we have devised to isolate cross-linked peptide-oligonucleotide complexes should be of general applicability and should facilitate future structure/function studies on other nucleic acid-binding proteins.

Amino Acids↗
PubMedDate not supplied

Serum autoantibodies in systemic lupus erythematosus and correlation with cutaneous features.

Eighty-eight untreated systemic lupus erythematosus patients collected from rheumatology services were studied for the presence of discoid, molar and vasculitic lesions and photosensitivity. Their sera were examined for antibodies to native deoxyribonucleic acid (nDNA), nuclear ribonuclear protein (nRNP), the non-nucleic acid nuclear macromolecule Sm, the nuclear and cytoplasmic macromolecules La(SSB) and the predominantly cytoplasmic macromolecule Ro(SSA). These studies demonstrated that 42% of the lupus patients possessed nDNA; 36% nRNP; 24% Ro(SSA) and 9% anti La(SSB) antibodies. Six patients failed to demonstrate significant antinuclear antibody titers (mouse liver substrate), and 4 of these possessed anti Ro(SSA) antibodies. Correlation to the cutaneous lesions among this lupus population possessing various serum antibodies revealed a trend toward photosensitivity and molar dermatitis in the Ro(SSA), La(SSB), Ro/la and the ANA negative, ANA negative/Ro positive groups of lupus patients.

Antibodies, Antinuclear↗