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PubMed1983

Lack of single-strand DNA-binding protein amplification under conditions of SOS induction in E. coli.

A two site immunoradiometric assay (IRMA) for quantification of the recA protein has been recently described (Paoletti et al. 1982). We have used a similar technique to monitor the possible amplification of the ssb protein in E. coli after induction of the SOS repair process by various DNA damaging agents. Under these conditions, while we have been able to detect a full amplification of recA protein, we failed to observed any amplification of the ssb protein.

Bacterial Proteins↗

PubMed1984

RecA protein--promoted lambda repressor cleavage: complementation between RecA441 and RecA430 proteins in vitro.

Induction of prophage lambda occurs in recA441 mutant lysogens after a shift to 42 degrees C in the presence of adenine. If the synthesis of RecA441 protein is maintained at a low basal level by the presence of a second mutation in the recA441 gene, recA453, induction of prophage lambda is prevented. The ability to induce prophage lambda is restored by the introduction, on a transducing phage, of a second recA gene carrying the recA430 mutation; by itself, the RecA430 protein is devoid of activity against the lambda repressor (Rebollo et al. 1984). In order to explain how the RecA430 protein might complement the RecA441 protein to provide lambda repressor cleavage in a recA453-441 (recA430) diploid lysogen, we characterized the cleavage reaction catalysed by a mixture of these proteins in vitro. Our results suggest that, in the presence of dATP, the RecA441 and RecA430 proteins form mixed multimers on single-stranded DNA, in which the RecA441 protein molecules enhance the DNA binding affinity of RecA430 protein molecules, but RecA430 protein molecules support no cleavage of the lambda repressor. Although the effects of the RecA430 and single-strand binding (SSB) proteins are similar in vitro, we show that the SSB protein cannot substitute for the RecA430 protein in restoring lambda repressor cleavage in a recA453-441 lysogen. Comparison of the stimulatory effect of long single-stranded DNA with that of (dA)14 oligonucleotides on the RecA441 protein-directed cleavage of the lambda repressor in the presence of various nucleoside triphosphates (NTPs) indicates that the cooperative binding of the RecA441 protein to single-stranded DNA stabilizes the RecA protein-DNA complexes so that they remain intact long enough to support cleavage of the lambda repressor. We conclude that the low basal level of the RecA441 protein in a recA453-441 cell is sufficient to cleave the lambda repressor, under conditions where a normal basal level of RecA430 protein is also present allowing the formation of mixed multimers on single-stranded DNA regions normally present in the cell.

Bacteriophage lambda↗

PubMed1993

Detection of DNA single-strand breaks in lymphocytes of smokers.

In a controlled study, ten male volunteers (five smokers and five nonsmokers) were subjected to different smoking conditions and compared to five nonsmokers, not exposed to cigarette smoke. During the 4 days of the study, nonsmoking periods were strictly controlled. On the first day the ten subjects were sham exposed. On the second day the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the environmental tobacco smoke. After another day of sham exposure the smoke exposure was repeated under the same conditions. Blood was drawn before and after exposure and DNA single-strand breaks (SSBs) were analyzed in lymphocytes immediately (1 h) after isolation of cells and after 4 h incubation at 37 degrees C, using a modified assay based on the nick translation reaction. Base levels of unscheduled DNA synthesis (UDS) and UDS levels were determined after 1 h incubation with methyl methanesulfonate. Duplicate analysis using the same method was performed in a second laboratory after transportation of blood samples at 0 degree C on a train from Munich to Hamburg. Tobacco smoke exposure of the subjects increased COHb and plasma cotinine levels. SSBs could be detected in all probands with some interindividual day-to-day and morning-to-evening variations. In four of five active smokers, SSB increases were found after smoking. In nonsmokers exposed to tobacco smoke no exposure-related variation in SSB levels could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1985

Effect of trimebutine on colonic myoelectrical activity in IBS patients.

The effect of trimebutine 100 mg i.v. and placebo on colonic myoelectrical activity was investigated in 10 patients with the irritable bowel syndrome (IBS) (5 constipated and 5 diarrhoeic), using an intraluminal probe supporting 8 groups of electrodes. At each site examined from transverse to sigmoid colon, the electromyograms exhibited two kinds of spike bursts: short spike bursts (SSB) localized at one electrode site and appearing rhythmically at 10.3/min, and long spike bursts (LSB), isolated or propagated orally or aborally. Computerized analysis of the duration of each kind of spike burst showed that, as compared to the control, trimebutine 100 mg, selectively inhibited by 43 to 73% the mean duration of LSB activity in the transverse, descending and sigmoid colon, from 0 to 30 min after administration. The inhibitory effect was similar in constipated and diarrhoeic patients. Placebo injection did not significantly affect (p greater than 0.05) the duration of LSB and SSB activity. Variance analysis indicated that the inhibitory effect of trimebutine was significantly greater (p less than 0.05) on LSB activity in the transverse than the descending colon, and that it was absent from the sigmoid colon. The results suggest that trimebutine selectively inhibits the propulsive activity of the proximal two thirds of the colon in IBS patients, and that this effect cannot entirely explain its therapeutic efficacy in IBS.

Adult↗

PubMed1995

The quality of DNA double-strand breaks: a Monte Carlo simulation of the end-structure of strand breaks produced by protons and alpha particles.

The quality of DNA damage induced by protons and alpha-particles of various linear energy transfer (LET) was studied. The aim was to single out specific lesions in the DNA molecule that might lead to biological endpoints such as inactivation. A DNA model coupled with a track structure code (MOCA-15) were used to simulate the lesions induced on the two helixes. Four categories of DNA breaks were considered: single-strand breaks (ssb), blunt-ended double-strand breaks (dsb, with no or few overlapping bases), sticky-ended double-strand breaks (with cohesive free ends of many bases), and deletions (complex lesions which involve at least two dsb within a small number of base pairs). Calculations were carried out assuming various sets of parameters characterizing the production of these different DNA breaks. No large variations in the yields of ssb and blunt- or sticky-ended dsb were found in the LET range between 10 and 200 keV/mu m. On the other hand, the yield of deletions increases up to about 100 keV/mu m and seems to reach a plateau at higher LET values. In the LET interval from 30 to 60 keV/mu m, protons proved to be more efficient than alpha-particles in inducing deletions. The induction of these complex lesions is thus dependent not simply on LET but also on the characteristics of the track structure. Comparison with RBE values for cell killing shows that this special class of dsb might play an important role in radiation-induced cell inactivation.

Alpha Particles↗

PubMed1994

In vitro reconstitution of homologous recombination reactions.

The proteins essential to homologous recombination in E. coli have been purified and their individual activities have been identified, permitting biochemical reconstitution of steps that comprise the cellular recombination process. This review focuses on the biochemical events responsible for the initiation and homologous pairing steps of genetic recombination. The properties of an in vitro recombination reaction that requires the concerted action of recA, recBCD, and SSB proteins and that is stimulated by the recombination hotspot, Chi(chi), are described. The recBCD enzyme serves as the initiator of this reaction; its DNA helicase activity produces single-stranded DNA that is used by the recA protein to promote homologous pairing and DNA strand invasion of supercoiled (recipient) DNA. The SSB protein acts to trap the single-stranded DNA produced by recBCD enzyme and to facilitate pairing by the recA protein. The chi regulatory sequence acts in cis by attenuating the nuclease, but not the helicase, activity of recBCD enzyme. This attenuation assures the preservation of ssDNA produced by the DNA helicase activity and is responsible for the simulation in vitro and, presumably, in vivo. The attenuation of nuclease activity by chi results in the loss or functional inactivation of the recD subunit.

DNA↗

PubMed1993

Evaluation of DNA damage by alkaline elution technique after inhalation exposure of rats and mice to 1,3-butadiene.

The alkaline filter elution technique was used to evaluate single strand breaks (SSB), DNA-DNA (DDCL) and DNA-protein cross-links (DPCL) in liver and lung of male rats (Sprague-Dawley) and male mice (B6C3F1) after exposure to 2000 ppm 1,3-butadiene (BD) for 7 days (7 h/day and/or to 100, 250, 500, 1000) 2000 ppm BD for 7 h. SSB were detected in liver DNA of both species at 2000 ppm. Cross-links are more pronounced in mouse lung than in mouse liver. Elution rates of lung DNA from mice exposed for 7 h to different concentrations of BD revealed an increase in cross-links between 250 and 500 ppm, and a further increase in cross-links up to 2000 ppm. No such signs of genotoxicity could be observed for the lung of rats. Our data support the involvement of reactive metabolites (epoxybutene and especially diepoxybutane) in butadiene-induced carcinogenesis in the mouse but not to that extent in the rat.

Animals↗

PubMed1992

DNA polymerase delta and epsilon holoenzymes from calf thymus.

Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) delta completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (or E. coli single-strand DNA binding protein, SSB). Pol epsilon core alone is able to synthesize the products on singly-primed ssDNA. However, DNA synthesis by pol epsilon was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB. This stimulation of pol epsilon by PCNA/RF-C/SSB appears to be the superposition of two events: pol epsilon holoenzyme (pol epsilon, PCNA, RF-C) synthesized longer products than its pol epsilon core counterpart, but elongated less primers. Furthermore, we analyzed the cooperative action of pol alpha/primase with pol delta or pol epsilon holoenzymes on unprimed M13 DNA. While pol delta displayed higher dNMP incorporation than pol epsilon, when a single primer was preannealed to DNA, pol epsilon was more efficient in the utilization of the primers synthesized by pol alpha/primase. Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol epsilon holoenzyme, than for pol delta. Our data support the hypothesis of pol delta as the leading and pol epsilon as the second lagging strand replication enzyme.

Animals↗

PubMed1990

The influence of Na+,K(+)-pump blockade on doxorubicin-mediated cytotoxicity and DNA strand breakage in human tumor cells.

We have previously shown that blockade of the Na+,K(+)-pump by the cardiac glycoside ouabain produces doxorubicin resistance and decreases topoisomerase II-mediated DNA strand breakage in hamster cells. To determine if this were a general phenomenon, the effect of pump blockade on doxorubicin resistance was assessed in three human tumor cell lines: A549 lung and HT29 colon adenocarcinomas and U1 melanoma. When cells were exposed to 1 microM ouabain prior to and during incubation with doxorubicin, cytotoxicity was markedly reduced. Ouabain had no effect on either the influx or the efflux of doxorubicin. However, all cell lines showed a ouabain-induced decrease in doxorubicin-induced topoisomerase-mediated DNA strand breakage (SSB). These data suggest that blockade of the Na+,K+ pump decreases doxorubicin cytotoxicity in human tumor cells by inhibiting topoisomerase-mediated SSB. Furthermore, they indicate that altered ionic gradients are a potential cause of resistance to drugs that use topoisomerase II as a target.

Adenocarcinoma↗

PubMed1998

Induction of single-strand breaks in lymphocyte DNA of rats exposed to hyperoxia.

A modified technique of alkaline filter elution was used to evaluate single-strand breaks (SSB) as a measure of DNA lesions in lymphocytes of male Wistar rats exposed to 60 or 80 vol% oxygen, over 23 h/day and for 6 or 5 days, respectively. An acceleration of elution was observed with samples from experiments at both increased levels of oxygen. With proteinase K treatment, a twofold increase in elution rates after exposure to 60 vol% oxygen and a threefold increase at 80 vol% oxygen were indicative of increased occurrence of SSB. This is explained by an involvement of reactive oxygen species in DNA damage under elevated oxygen levels.

Animals↗

PubMed1996

Differential single- versus double-strand DNA breakage produced by doxorubicin and its morpholinyl analogues.

The morpholinyl analogues of doxorubicin (DOX) have previously been reported to be non-cross-resistant in multidrug resistant (MDR) cells due to a lower affinity for P-glycoprotein relative to the parent compound. In order to further investigate the mechanisms of action of these morpholinyl anthracyclines, we examined their ability to cause DNA single- and double-strand breaks (SSB, DSB) and their interactions with topoisomerases. Alkaline elution curves were determined after 2-h drug treatment at 0.5, 2 and 5 microM, while neutral elution was conducted at 5, 10 and 25 microM in a human ovarian cell line, ES-2. A pulse-field gel electrophoresis assay was used to confirm the neutral elution data under the same conditions. Further, K-SDS precipitation and topoisomerase drug inhibition assays were used to determine the effects of DOX and the morpholinyl analogues on topoisomerase (Topo) I and II. Under deproteinated elution conditions (pH 12.1), DOX, morpholinyl DOX (MRA), methoxy-morpholinyl DOX (MMDX) and morpholinyl oxaunomycin (MX2) were equipotent at causing SSB in the human ovarian carcinoma cell line, ES-2. However, neutral elution (pH 9.6) under deproteinated conditions revealed marked differences in the degree of DNA DSB. After 2-h drug exposures at 10 microM, DSBs were 3300 rad equivalents for MX2, 1500 for DOX and 400 for both MRA and MMDX in the ES-2 cell line. Pulse-field data substantiated these differences in DSBs, with breaks easily detected after MX2 and DOX treatment, but not with MRA and MMDX. DOX and MX2 thus cause DNA strand breaks selectively through interaction with Topo II, but not Topo I. In contrast, MRA and MMDX cause DNA breaks through interactions with both topoisomerases with a predominant inhibition of Topo I.

Antibiotics, Antineoplastic↗

PubMed2000

Rheumatoid factors in systemic lupus erythematosus: association with clinical and laboratory parameters. SLE study group.

The prevalence and clinical and laboratory associations of IgM, IgG and IgA rheumatoid factors (RF) were determined in 352 patients with systemic lupus erythematosus (SLE). IgM, IgG, and IgA class RF were detected in 17.9%, 20.5%, and 20.5% of the sera, respectively. RF were associated with sicca syndrome, hypergammaglobulinemia, high titer of antinuclear antibodies, anemia, SSA- and SSB-antibodies, and with the presence of HLA-DR3. RF correlated negatively with nephritis and livedo racemosa. Moreover, we observed an association of RF and parameters of inflammatory activity such as elevated erythrocyte sedimentation rate (ESR) and leukopenia. Analysis of immunoglobulin classes revealed that laboratory parameters of inflammatory activity, SSA- and SSB-antibodies and HLA-DR3 correlated with IgA RF only. IgA RF define a subgroup of SLE patients characterized by distinct autoimmune phenomena and high disease activity in the absence of nephritis.

Antibodies, Antinuclear↗

PubMed2002

Monte Carlo simulation of strand-break induction on plasmid DNA in aqueous solution by monoenergetic electrons.

The radiation-induced process of strand breaks on pBR322 plasmid DNA in aqueous solution for different energy electrons was studied by Monte Carlo simulation. Assumptions of induction mechanisms of single- and double-strand breaks (SSBs and DSBs) used in the simulation are that SSB is induced by OH or H reaction with DNA and that DSB is induced by two SSBs on the opposite strands within 10 bp. Dose-response relationships of SSBs and DSBs were demonstrated for monoenergetic electrons of 100 eV, 10 keV, 1 keV and 1 MeV, and the yields of SSB and DSB were calculated. The dose-response relationships of SSBs and DSBs can be fitted by linear and linear-quadratic functions, respectively. The ratio of quadratic to linear components of DSB induction changes due to the electron energy. A high contribution of the linear component is observed for 1 keV electrons in the dose range below 160 Gy. The yields of SSBs and DSBs for all examined electron energies lie well within the experimental data when the probability of strand-break induction by OH and H is assumed to be around 0.1-0.2. The yield of SSBs has a minimum at 1 keV, while the yield of DSBs has a maximum at 1 keV in the examined energies. The strand breaks are formed most densely for 1 keV electrons.

Chromosome Breakage↗

PubMed2006

Effects of potato plants expressing a barley cystatin on the predatory bug Podisus maculiventris via herbivorous prey feeding on the plant.

The aim of this study was to assess the effects of potato plants expressing a barley cystatin on a potentially cystatin-susceptible natural enemy by predation on susceptible and non-susceptible preys feeding on the plant. We have focussed on the impact of the variant HvCPI-1 C68 --> G, in which the only cysteine residue was changed by a glycine, on the growth and digestive physiology of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the Egyptian cotton leafworm (ECW), Spodoptera littoralis. Moreover, we have studied the prey-mediated effects of the barley cystatin at the third trophic level, using the predatory spined soldier bug (SSB), Podisus maculiventris, as a model. Feeding trials conducted with CPB larvae reared on transgenic potato plants expressing the C68 --> G variant resulted in significantly lower weight gains compared to those fed on non-transformed (NT) plants. On the contrary, larger weight gains were obtained when ECW larvae, that lack digestive cysteine proteases, were reared on transgenic potato expressing the cystatin, as compared to larvae fed on NT plants. No negative effects on survival and growth were observed when SSB nymphs were exposed to HvCPI-1 C68 --> G by predation on either CPB or ECW larvae reared on transgenic potato plants expressing the barley cystatin, despite the fact that the inhibitor suppressed in vitro gut proteolysis of the predatory bug. To investigate the physiological background, biochemical analysis were carried out on guts of insects dissected at the end of the feeding assays.

Amino Acid Substitution↗

PubMed1990

Picogram quantitation of total DNA using DNA-binding proteins in a silicon sensor-based system.

We report a rapid and reproducible method to quantify total DNA at picogram levels. Two high-affinity DNA-binding proteins are used to construct a sandwich assay and a semiconductor sensor is used for quantitation. Single-stranded DNA-binding protein (SSB) from Escherichia coli is conjugated with a linker molecule, biotin, for specific capture of the DNA complex onto a membrane. Monoclonal anti-DNA antibody is conjugated with an enzyme, urease, for signal generation. To detect DNA, a sample is denatured to form single-stranded DNA and then incubated with a reagent containing both DNA-binding protein conjugates and streptavidin. After incubation of the reagent with the DNA sample for 1 h at 37 degrees C to form a complex of streptavidin--biotin--SSB--DNA--anti-DNA--urease, the mixture is filtered through a biotin-coated nitrocellulose membrane which binds the streptavidin component of the complex. The unbound reagent is washed off the membrane, and then the captured DNA complex is detected with a light-addressable potentiometric sensor which measures the pH change catalyzed by the urease in the complex. This assay can detect 2 pg of DNA with a quantitation coefficient of variation of less than 10% in the range 10 to 200 pg.

Animals↗

PubMed1991

Primary structure of the two variants of Xenopus laevis mtSSB, a mitochondrial DNA binding protein.

The primary structure of the single-stranded DNA binding protein from Xenopus laevis oocyte mitochondria (mtSSB) has been determined by Edman degradation of the intact molecule and peptides derived from partial alpha-chymotrypsin proteolysis and enzymatic cleavage with trypsin and endoproteinase Glu-C. The native mtSSB is composed of two related polypeptide chains, mtSSBs and mtSSBr. The sequence of mtSSBs consists of 129 amino acids with a calculated molecular mass of 14,627 Da. Comparison of the first 80 residues of the two chains reveals 91% identity. A high degree of similarity is found between mtSSB and Escherichia coli SSB or F sex factor SSB.

Amino Acid Sequence↗

PubMed1989

Developmental change and molecular properties of somatostatin receptors in the rat cerebral cortex.

The postnatal development and molecular properties of somatostatin receptor were studied in rat cerebral cortex. With [125I-Tyr11]SRIF as a radiolabeled ligand, the specific ligand binding to crude membrane increased transiently in the early phase of postnatal development and then decreased. This increase of somatostatin binding was mainly due to the increased number of binding sites. The two subtypes classified by Tran et al., SSA and SSB, were confirmed and the studies on the relative amount of the subtypes revealed that more SSA subtype was expressed compared with SSB subtype during a week after birth, but, thereafter, both subtypes were almost equally expressed throughout the developmental stages tested. Molecular weight of the covalently labeled somatostatin receptor (SSA subtype), which was determined with the aid of the cross-linking agents, was estimated to be approximately 71,000 with no intramolecular disulfide bond.

Aging↗

PubMed1987

Induction of DNA crosslinks in vitro upon reduction of the nitroimidazole-aziridines RSU-1069 and RSU-1131.

The interaction of the nitroimidazole-aziridines RSU-1069 and RSU-1131, as parent or radiation-reduced species, with plasmid DNA in aqueous solution at pH 7 results in strand breakage. The yields of DNA single strand breaks (ssb), "alkali-labile" damage and DNA crosslinks induced by these alkylating agents have been assessed. It is shown that DNA crosslinks are induced only by the reduced nitro-compounds. RSU-1069, as parent or reduced compound, is more efficient at producing these effects than the equivalent form of RSU-1131. Further, RSU-1069 is about 2 X more susceptible to nucleophilic attack by inorganic phosphate and deoxynucleotides than RSU-1131. RSU-1069 also shows greater selectivity for reaction with the nucleotide base moiety than does the less-reactive monomethyl analogue, RSU-1131. The yields of ssb and "alkali-labile" damaged sites induced by the two agents reflect their respective chemical reactivities and appear largely to determine their aerobic cytotoxicities. In contrast, the yield of DNA crosslinks induced by the reduced compounds appears to correspond rather better with the observed hypoxic cytotoxicities. From these findings it is suggested that the induction of DNA crosslinks by these agents may play a major role in their effectiveness as hypoxia-selective cytotoxins.

DNA↗