Hydrogen peroxide-induced base damage in deoxyribonucleic acid

Aqueous solutions of calf thymus deoxyribonucleic acid (DNA) were exposed to hydrogen peroxide in the presence of air. Base products formed in DNA were identified and quantitated following acid hydrolysis and trimethylsilylation using gas chromatography-mass spectrometry. The yields of these products were dependent upon the hydrogen peroxide concentration, and increased in the following order: 8-hydroxyadenine, cytosine glycol, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine, thymine glycol, and 4,6-diamino-5-formamidopyrimidine. Previous studies have shown that these compounds are typically formed in DNA in aqueous solution by hydroxyl radicals generated by ionizing radiation. Hydrogen peroxide is thought to participate in a Fenton-like reaction with transition metals, which are readily bound to DNA in trace quantities, resulting in the production of hydroxyl radicals close to the DNA. This proposed mechanism was examined by exposing DNA to hydrogen peroxide either in the presence of a hydroxyl radical scavenger or following pretreatment of DNA with metal-ion chelators. The results indicate that trace quantities of transition metal ions can react readily with hydrogen peroxide to produce radical species. The production of radical species was monitored by determining the altered bases that resulted from the reaction between radicals and DNA. The yields of the base products were reduced by 40 to 60% with 10 mmol dm-3 of dimethyl sulfoxide. A 100-fold increase in the concentration of dimethyl sulfoxide did not result in a further reduction in hydrogen peroxide-induced base damage. DNA which was freed from bound metal ions by pretreatment with metal ion chelators followed by exhaustive dialysis was found to be an ineffective substrate for hydrogen peroxide. The yields of base products measured in this DNA were at background levels. These results support the role of metal ions bound to DNA in the site-specific formation of highly reactive radical species, most likely hydroxyl radicals, in hydrogen peroxide-induced damage to the bases in DNA.     

Monoamine and amino acid content in brain regions of Brattleboro rats

Monoamine and amino acid content were measured in brain regions from 12 week old male, homozygous Brattleboro (DI,n=12) and Long-Evans control (LE,n=12) rats. Norepinephrine (NE) content was significantly elevated (16–25%) in the spinal cord, pons-medulla and anterior hypothalamus of DI rats when compared to LE controls. NE content of the neurointermediate lobe of pituitary in DI rats was almost twice that of LE controls. Serotonin content was also significantly elevated in the spinal cord, pons-medulla, anterior hypothalamus and forebrain of DI rats relative to the LE controls. Taurine content in DI rats was increased (31–42%) above that of LE rats in the anterior hypothalamus, striatum and forebrain. Glutamine content was also greater in DI rats than LE in the spinal cord, pons-medulla, anterior hypothalamus, striatum, hippocampus and forebrain. The changes in monoamine and amino acid content were discussed in relation to the cardiovascular and osmoregulatory deficits that are present in DI rats due to arginine vasopressin (AVP) deficiency. The possible role of AVP in modulating NE turnover was also discussed. The increase in brain TAU content in DI rats may be a physiological response to hypernatremia.     

Sex hormone-dependent brain maturation and sexual behaviour in rats.

In male and female rats the endogenous steroid and gonadotrophin secretion was inhibited by injecting high doses of chlormadinone acetate (CmAc) from day 14 to 24 of life, i. e. during the period of brain maturation. In adulthood the males treated prepubertally with CmAc exhibited reduced sexual activity and fertility, whereas the females did not differ from the controls. More complete sex hormone deficiency during brain maturation was achieved by castration on day 14 of life. Controls were castrated at normal puberty time (40--60 days). Both groups were then substituted with androgens or oestrogens. In the females castrated on day 14 no impairment of sexual behaviour was observed as compared to the later castrated controls. In contrast, the early castrated males showed delayed onset of mounting behaviour. At autopsy, the weights of their sex organs were found to be lower than in the controls despite equal testosterone replacement for several months. These findings speak in favour of a permanently diminished responsiveness to androgens in males having been exposed to more or less severe androgen deficiency during sex specific brain maturation. Hence, the maturation of a male hypothalamus as well as the differentiation appears to depend at least in part on the presence of androgens, whereas in females it runs without hormonal influence.     

Quantitative determination of deoxyribonucleic acid in rat brain

1. A procedure is given for spectrophotometric analysis of rat brain DNA after its resolution into component bases. Amounts of tissue in the range 50-100mg. can be used. 2. The amount of DNA obtained by the present method is 80% greater than that reported for rat brain by a previous procedure specific for DNA thymine. Identity of the material is established by the base ratios of purines and pyrimidines. The features responsible for the higher yield are the presence of dioxan during alkaline hydrolysis of tissue, the determination of the optimum concentration of potassium hydroxide in this step and omission of organic washes of the initial acid-precipitated residues. 3. The requirement for dioxan during alkaline hydrolysis suggests a possible association of brain DNA with lipid. The concentration of potassium hydroxide that gives maximum yield is 0.1m, indicating that there may be internucleotide linkages in this DNA that are more sensitive to alkali than those of liver or thymus DNA. 4. This procedure gives low yields of DNA from liver. It is not suitable for analysis of the DNA from this tissue.     

Analysis and sorting of living cells according to deoxyribonucleic acid content.

The methods for measuring the deoxyribonucleic acid content of individual mammalian cells and sorting them on the basis of this parameter have until now required fixation or other treatment which renders the cells nonviable. Using a class of bis-benzimidazole dyes, Hoechst 33258 and 33342 and a multiparameter computer-controlled cell sorter, we have been able to stain and separate living cells in the G1, S, and G2+M phases of the cell cycle and to continue their growth in tissue culture with high retention of viability (greater than 90%) and no increase in heteroploidy. The quenching of the fluorescence of the bound dye by 5-bromodeoxyuridine incorporated into cellular deoxyribonucleic acid is being used with the flow system to detect and isolate mutants in deoxyribonucleic acid metabolism spectroscopically.     

Some applications of deoxyribonucleic acid base composition in bacterial taxonomy

The melting pointT _m, the mean molar (guanine+cytosine) composition and the compositional distribution of purified DNA from several strains ofXanthomonas, Chromobacterium and yellow-pigmented marine bacteria have been determined. These groups were selected because they had been analyzed adansonially. Ten strains ofXanthomonas had an average molar (guanine+cytosine) composition within the range 66.0–68.2%, which was very close to that ofPseudomonas (60–68%), as expected. All strains ofChromobacterium (six of theviolaceum biotype and three of thelividum biotype) had mean molar (guanine+cytosine) compositions within the range 63.4–71.4%. The yellow-pigmented marine flavobacteria had mean molar (guanine+cytosine) compositions of 35.6–40.6%. This suggests that they would not be genetically related to the yellow-pigmentedXanthomonas, nor to facultative aerobic organisms, such asAeromonas and theEnterobacteriaceae. The yellow-pigmented marine swarming bacteria, which resembleCytophaga, fell into two separate groups: some had a mean molar (guanine+cytosine) composition of about 34%, others were around 63%. This suggests genetic heterogeneity. The compositional distribution of DNA molecules was on the whole more narrow in polarly flagellated than in peritrichous organisms.     

The activity of deoxyribonucleic acid polymerase and deoxyribonucleic acid synthesis in nuclei from brain fractionated by zonal centrifugation

1. The DNA polymerase (EC 2.7.7.7) activity in purified intact brain nuclei from infant rats was investigated. The effects of pH, Mg(2+), glycerol, sonication and storage of the nuclei under different conditions were examined and a suitable assay system was established. 2. The nuclei from infant brain cells were fractionated by zonal centrifugation in a discontinuous sucrose gradient into five zones: zone (I) contained neuronal nuclei (59%) and astrocytic nuclei (41%); zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (19%); zone (III) contained astrocytic nuclei (82%) and oligodendrocytic nuclei (18%); zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained oligodendrocytic nuclei (100%). 3. The content of DNA, RNA and protein for each fraction was measured. 4. The distribution of DNA polymerase activity in the fractionated infant and adult rat brain nuclei was determined. The highest activity was found in the neuronal nuclei from zone (I) and the following zones exhibited a progressive decline. In contrast with the nuclei from infant rats those from adults had a much higher activity and expressed a preference for native DNA as template. 5. The deoxyribonuclease activity in all classes of nuclei was measured with [(3)H]DNA as substrate. A general correspondence in the pattern of the relative activities in the nuclear fractions with the distribution of DNA polymerase was found. 6. The incorporation of [(3)H]thymidine into nuclear DNA in infant and adult rat brain was investigated. The specific radioactivity of the DNA in the 10-day-old rats was highest in zone (V) whereas in the nuclei of adult rats, which exhibited a comparatively low incorporation, the highest specific radioactivity was associated with zones (I) and (V).     

C14-variamycin content in normal and tumorous brain tissues of white rats]

Penetration of variamycin, a new antitumor antibiotic into the normal and tumor tissues of the brain of rats with multiform glioblastoma was investigated. The content of the C14-labeled antibiotic was determined radiometrically. The radioactive label penetrated into the normal and tumor tissues of the brain during the first hours after the drug administration. The level of the radioactivity in the tumor tissue was higher than that in the normal brain tissue during the whole period of the study. The greatest deviation in the contents of the radioactive label in the tumor and normal tissues was observed 2 and 3 hours after administration of the labeled antibiotic, i. e. 4.3 and 3.6 times respectively.     

Involvement of deoxyribonucleic acid polymerase beta in nuclear deoxyribonucleic acid synthesis.

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.