Scission Products Research Articles

DNA lability induced by nimustine and ramustine in rat glioma cells.

The DNA labile sites induced by two nitrosoureas, nimustine (ACNU) and ramustine (MCNU) synthesised in Japan, have been examined in highly reiterated DNA sequences of rat glioma cells. Reiterated fragments of 167 and 203 base pairs (bp), obtained after Hind III and Hae III restriction endonuclease digestion of rat glioma cells DNA, were used as target DNA sequences to determine the labile sites. In vitro reaction with ACNU and MCNU resulted in scission products corresponding to the locations of guanine. Subsequent piperidine hydrolysis produced more frequent breaks of the phosphodiester bonds at guanine positions, thus forming alkali-labile sites.

Wavelength Dependence for Human Redoxy-Endonuclease-Mediated DNA Cleavage at Sites of UV-Induced Photoproducts

Redoxy-endonuclease, an enzyme present in human and other cells, recognizes monobasic photoproducts that occur primarily at sites of cytosine following UV-irradiation of DNA at 254 nm. The wavelength dependence for formation of these photoproducts was determined using end-labeled DNA fragments of defined sequence irradiated with monochromatic light ranging from 254-360 nm as substrates for redoxy-endonuclease partially purified from HeLa cells. The base specificity and extent of DNA cleavage were determined by analysis of the enzyme-generated DNA scission products on DNA sequencing gels. Maximal incision at sites of cytosine and thymine was observed at 280 nm, suggesting that these photoproducts may be relevant with respect to the biological effects of solar radiation.

Stabilization of polypropylene to γ-initiated oxidation

The chemical and physical property changes produced in polypropylene samples by γ-irradiation in air have been measured for a range of potential stabilizers. Impact properties of post-irradiation aged samples were compared with the yields of tertiary and secondary hydroperoxides, peroxyl radicals and ketones. Some additives markedly increased the initial rate of peroxyl radical decay, but this effect did not correlate with subsequent stability. Retention of impact strength was found for additives which minimized the initial formation of hydroperoxides and ketonic, backbone scission products. A piperidyl additive gave good protection but was especially effective when used in combination with some benzophenone derivatives. The latter were largely ineffective radiation stabilizers when used alone. None of the stabilizers appeared to reduce the initial yield of peroxyl radicals produced in the γ-irradiated polymer and the piperidyl compound was only converted to its nitroxide during the post-irradiation period.

Oxyhydrative scission of olefins: III. An extension to various olefins and ketones

In an attempt to extend the oxyhydrative scission reaction which is typical with n-olefins, three olefins and four ketones with various structures have been subjected to oxidation over a V 2O 5-MoO 3 ( V Mo = 9 1 ) catalyst in the presence of water vapor. Styrene was selectively oxidized at 453–533 K to benzaldehyde and benzoic acid in conformity with the proposed scission scheme. Cyclohexene and l-chloro-2-butene, however, were subjected to dehydrogenation and dehydrochlorination, respectively, at 423–533 K in preference to the scission reaction. Methylvinylketone underwent scission reaction to form acetic acid and carbon oxides at 423–473 K. 2-Chloro-3-butanone showed high reactivity even at 393 K and produced acetic acid very selectively; one of the expected scission products, acetylchloride, was hydrolyzed to acetic acid. Cycloketones, i.e., cyclopentanone and cyclohexanone, however, were oxidized only to carbon oxides. On the basis of these results the oxyhydrative scission mechanism and the requirements of reactants for it are discussed.

Ozonation of hydrocarbon diene elastomers: A mechanistic study

AbstractA study of the effects of ozonation on polybutadiene, polyisoprene, and several related hydrocarbon elastomers has shown that elastomers containing di‐substituted double bonds (e.g., cis‐1,4‐polybutadiene) give crosslinked products as well as chain scission products in nonpolar solvents, whereas those containing tri‐substituted double bonds (e.g., cis‐1,4‐polyisoprene) give chain scission products only. Both types of elastomer, however, give only chain scission products in polar solvents. Further investigation of the ozonation of elastomers, including the effect of ozonides of monoolefins and the solvent effect has led us to postulate that the chain scission involves the attack of a second ozone molecule on the preformed ozonide, and, the crosslinking is due to the attack of the biradical carbonyl oxide on the rubber.

Flow‐induced chain fracture of isolated linear macromolecules in solution

AbstractThe recently developed elongational flow technique is applied to the examination of flow‐induced chain rupture of macromolecules in solution. For both closely monodisperse atactic polystyrene (a‐PS) and poly(ethylene oxide) (PEO), the critical strain rate for extension (\documentclass{article}\pagestyle{empty}\begin{document}$ \dot \varepsilon _c $\end{document}) is found to depend upon molecular weight M as \documentclass{article}\pagestyle{empty}\begin{document}$ \dot \varepsilon _c \simeq M^{ - 1.5} $\end{document}, consistent with ideal Zimm dynamics. When the chains are subjected to strain rates beyond \documentclass{article}\pagestyle{empty}\begin{document}$ \dot \varepsilon _f $\end{document} the scission products correspond closely to one‐half of the initial molecular weight. The critical fracture stress depends upon molecular weight as \documentclass{article}\pagestyle{empty}\begin{document}$ \dot \varepsilon _f \simeq M^{ - 2} $\end{document}, enabling the prediction of the ultimate chain length which can be extended without fracture (\documentclass{article}\pagestyle{empty}\begin{document}$ \dot \varepsilon _c = \dot \varepsilon _f $\end{document}). For a‐PS this corresponds to M = 3 × 107. These findings are well accounted for by Stokes' Law applied to an extended bead–rod model. The calculated flow‐induced force in the chain corresponds closely to the rupture force of a covalent backbone bond calculated from a modified Arrhenius rate equation. During the prefracture stage (\documentclass{article}\pagestyle{empty}\begin{document}$ \dot \varepsilon _f > \dot \varepsilon > \dot \varepsilon _c $\end{document}) a‐PS shows anomalies in the flow‐induced birefringence, which suggest that the Phenyl side groups are becoming reoriented due to the progressive increase in free volume as the chemical backbone bonds stretch and the bond angles open.

Surface chemistry of alkylamines. 2. Methylamine and trimethylamine on W(100), W(100)-(5 .times. 1)-C and W(100)-(2 .times. 1)-O

The adsorption and reaction of mono- and trimethylamine have been investigated on W(100), W(100)-(5 x 1)-C, and W(100)-(2 x 1)-O, using temperature-programmed reaction spectroscopy. Reactivity and selectivity are dependent on the presence of the oxide or carbide overlayers and the degree of substitution of the amine. The activation energy for irreversible C-H bond cleavage in trimethyl- and methylamine was significantly greater on the -(2 x 1)-O and -(5 x 1)-C surfaces compared to W(100). Most notable was the elevated temperature up to where molecular amine desorption is observed on the -(2 x 1)-O: 650 and 600 K for methyl- and trimethylamine, respectively, corresponding to approximate desorption energies of 39 and 35 kcal/mol. The extent of amine substitution also qualitatively alters the product distribution, with CH/sub 4/ produced only from (CH/sub 3/)/sub 3/N and NH/sub 3/ only from (CH/sub 3/)NH/sub 2/ on W(100) and W(100)-(5 x 1)-C> No C-N bond scission products were observed on W(100)-(2 x 1)-O, consistent with the predominance of reversible molecular adsorption without significant decomposition. Possible reaction mechanisms are discussed in terms of competing C-H, N-H, and C-N bond activation.

Surface chemistry of alkylamines. 1. Ethylamine and triethylamine on W(100), W(100)-(5 .times. 1)-C, and W(100)-(2 .times. 1)-O

The adsorption and reaction of mono- and trimethylamine have been investigated on W(100), W(100)-(5 x 1)-C, and W(100)-(2 x 1)-O, using temperature-programmed reaction spectroscopy. Reactivity and selectivity are dependent on the presence of the oxide or carbide overlayers and the degree of substitution of the amine. The activation energy for irreversible C-H bond cleavage in trimethyl- and methylamine was significantly greater on the -(2 x 1)-O and -(5 x 1)-C surfaces compared to W(100). Most notable was the elevated temperature up to where molecular amine desorption is observed on the -(2 x 1)-O: 650 and 600 K for methyl- and trimethylamine, respectively, corresponding to approximate desorption energies of 39 and 35 kcal/mol. The extent of amine substitution also qualitatively alters the product distribution, with CH/sub 4/ produced only from (CH/sub 3/)/sub 3/N and NH/sub 3/ only from (CH/sub 3/)NH/sub 2/ on W(100) and W(100)-(5 x 1)-C> No C-N bond scission products were observed on W(100)-(2 x 1)-O, consistent with the predominance of reversible molecular adsorption without significant decomposition. Possible reaction mechanisms are discussed in terms of competing C-H, N-H, and C-N bond activation.

Quantitative microstructure determination of diene polymers by means of metathesis degradation

In the case of polybutadienes and butadiene-styrene copolymers, metathetical chain scission makes possible extensive microstructure characterization. The basic requirement of course is, besides the use of a highly selective catalyst system of sufficiently lasting activity, a knowledge of the degradation behaviour of the polymers. The most important results obtained are: (1) the discovery and the determination of the respective position of the chain-degradation and propagation equilibrium, as well as rate of its establishment; (2) the discovery that products having 1,2-butadiene units distributed blockwise show an aggravated degradation behaviour; (3) a reduction in the number of scission products which are generated from one chain segment, by shifting the reaction towards the thermodynamically stable cyclohexene compounds. Quantitative chirality determination of 1,2-butadiene and styrene dyads are presented.

Substrate specificity of a mammalian DNA repair endonuclease that recognizes oxidative base damage.

The substrate specificity of a calf thymus endonuclease on DNA damaged by UV ligh, ionizing radiation, and oxidizing agents was investigated. End-labeled DNA fragments of defined sequence were used as substrates, and the enzyme-generated scission products were analyzed by using DNA sequencing methodologies. The enzyme was shown to incise damaged DNA at pyrimidine sites. The enzyme incised DNA damaged with UV light, ionizing radiation, osmium tetroxide, potassium permanganate, and hydrogen peroxide at cytosine and thymine sites. The substrate specificity of the calf thymus endonuclease was compared to that of Escherichia coli endonuclease III. Similar pyrimidine base damage specificities were found for both enzymes. These results define a highly conserved class of enzymes present in both procaryotes and eucaryotes that may mediate an important role in the repair of oxidative DNA damage.

Substrate specificity of a mammalian DNA repair endonuclease that recognizes oxidative base damage.

The substrate specificity of a calf thymus endonuclease on DNA damaged by UV ligh, ionizing radiation, and oxidizing agents was investigated. End-labeled DNA fragments of defined sequence were used as substrates, and the enzyme-generated scission products were analyzed by using DNA sequencing methodologies. The enzyme was shown to incise damaged DNA at pyrimidine sites. The enzyme incised DNA damaged with UV light, ionizing radiation, osmium tetroxide, potassium permanganate, and hydrogen peroxide at cytosine and thymine sites. The substrate specificity of the calf thymus endonuclease was compared to that of Escherichia coli endonuclease III. Similar pyrimidine base damage specificities were found for both enzymes. These results define a highly conserved class of enzymes present in both procaryotes and eucaryotes that may mediate an important role in the repair of oxidative DNA damage.

Mechanism of action of a mammalian DNA repair endonuclease

The mechanism of action of a DNA repair endonuclease isolated from calf thymus was determined. The calf thymus endonuclease possesses a substrate specificity nearly identical with that of Escherichia coli endonuclease III following DNA damage by high doses of UV light, osmium tetroxide, and other oxidizing agents. The calf thymus enzyme incises damaged DNA at sites of pyrimidines. A cytosine photoproduct was found to be the primary monobasic UV adduct. The calf thymus endonuclease and E. coli endonuclease III were found to possess similar, but not identical, DNA incision mechanisms. The mechanism of action of the calf thymus endonuclease was deduced by analysis of the 3' and 5' termini of the enzyme-generated DNA scission products with DNA sequencing methodologies and HPLC analysis of the material released by the enzyme following DNA damage. The calf thymus endonuclease removes UV light and osmium tetroxide damaged bases via an N-glycosylase activity followed by a 3' apurinic/apyrimidinic (AP) endonuclease activity. The calf thymus endonuclease also possesses a novel 5' AP endonuclease activity not possessed by endonuclease III. The product of this three-step mechanism is a nucleoside-free site flanked by 3'-and 5'-terminal phosphate groups. These results indicate the conservation of both substrate specificity and mechanism of action in the enzymatic removal of oxidative base damage between prokaryotes and eukaryotes. We propose the name redoxy endonucleases for this group of enzymes.

Analysis of products formed during bleomycin-mediated DNA degradation

By the use of DNA, copolymers of defined nucleotide composition, and a synthetic dodecanucleotide having putative bleomycin cleavage sites in proximity to the 5'- and 3'-termini, the products formed concomitant with DNA strand scission have been isolated and subjected to structural identification and quantitation via direct comparison with authentic synthetic samples. The products of DNA strand scission by Fe(II)-bleomycin include oligonucleotides having each of the four possible nucleoside 3'-(phosphoro-2''-O-glycolates) at their 3'-termini, as well as the four possible base propenals. At least for 3-(adenin-9'-yl)propenal and 3-(thymin-1'-yl)propenal, the products formed were exclusively of the trans configuration.

Volatiles from thermal decomposition of isomeric methyl (12S, 13S)‐(E)‐12,13‐epoxy‐9‐hydroperoxy‐10‐octadecenoates

AbstractTwo epimers of methyl (12S,13S)‐(E)‐12,13‐epoxy‐9‐hydroperoxy‐10‐octadecenoate were isolated after esterification of a mixture of fatty acids obtained from decomposition of (13S)‐(9Z,11E)‐13‐hydroperoxy‐9,11‐octadecadienoic acid by an Fe++‐cysteine catalyst. These epimeric epoxyhydro‐peroxyoctadecenoates were decomposed by heat (210 C) in the injection port of a gas chromatograph, and the cleavage fragments were subsequently separated by gas chromatography (GC) and identified by mass spectrometry (MS). Among the scission products obtained, the most prominent in the GC peak profile were methyl octanoate and methyl 9‐oxononanoate. Other peaks were identified as pentane, 1‐pentanol, hexanal, 2‐heptanone, 2‐pentylfuran, methyl heptanoate, 2‐octenal, 4,5‐epoxy‐2‐decenal, methyl 8‐(2‐furyl)‐octanoate, 11‐oxo‐9‐undecenoate and methyl 13‐oxo‐9,11‐tridecadienoate. In addition, 3,4‐epoxynonanal, methyl 8‐oxooctanoate, 3‐hydroxy‐2‐pentyl‐2,3‐dihydrofuran and methyl 10‐oxodecanoate were tentatively identified. Except for the furan compounds, the formation of the fragmentation products could be explained by conventional free‐radical scission mechanisms.

Cytochrome P-450 oxidation of alkanes originating as scission products during lipid peroxidation.

Alkanes of low molecular weight, as well as malondialdehyde, originate during lipid peroxidation. Ethane and pentane are the most prominent and are probably scission products of omega-3 and omega-6 unsaturated fatty acids, respectively. Measurement of exhaled alkanes is the most reliable procedure for determining lipid peroxidation in vivo. Alkanes appear in the breath of rats 15 min after administration of CBrCl3 i.p., and are also formed in small amounts endogenously. Alkanes exhaled from untreated rats in a closed system, in which CO2 is absorbed and O2 supplied, reached steady-state levels after different times, indicating that these volatile gases are metabolized at variable rates. Metabolism was verified by injecting alkanes into the closed system. Pentane was metabolized 5-10 times faster than ethane, and was species- and strain-dependent. Administration of drugs which inhibit or induce cytochrome P-450 indicated that a particular isoenzyme might be involved in the oxidation of small alkanes. SKF 525-A or benzoflavone did not inhibit, but tetrahydrofuran and ethanol were effective inhibitors. Inducing effects of phenobarbital, methylcholanthrene or ethanol were insignificant. Incubation of microsomes with NADPH and O2, either with or without Fe-ADP, to elicit lipid peroxidation confirmed the findings in vivo. Ethane and pentane were formed in similar quantities. Inhibition of alkane oxidation with CO or ethanol increased the amount of pentane three- to four-fold, indicating that inhibition of metabolism enhances alkane release. The ratio of unmetabolized pentane to ethane reflects the membrane ratio of omega-6 to omega-3 unsaturated acids. Different types of alkane release were observed following administration of paracetamol or CCl4 to mice, indicating differences in the peroxidative attack. CCl4 destroys cytochrome P-450 dose dependently, so that it loses its capacity to oxidize pentane, whereas paracetamol does not inactivate the mono-oxygenase. Monitoring the elimination rate of injected pentane is recommended as a reliable non-invasive procedure for testing the functional state of hepatic cytochrome P-450.

Effects of phenobarbitone and phenytoin on folate catabolism in the rat

The effects of phenobarbitone and phenytoin on the catabolism of oral [2- 14C] and [3′,5′,7,9- 3H] folic acid were investigated. Normal rats were found to excrete an excess of 3H-labelled compounds into the urine and 14C-labelled compounds into the faeces. Phenytoin abolished this urinary 3H imbalance and also delayed and prolonged the overall excretion of radioactive material. Phenobarbitone appeared to increase the amounts of urinary scission products in the first 24 hr but over the 0–72 hr period both anticonvulsants decreased folate polyglutamate catabolism. As the anticonvulsants used in these experiments decreased folate catabolism in the rat it is unlikely that the megaloblastic anaemia caused by chronic anticonvulsant therapy is due to induction of the enzymes responsible for folate breakdown in vivo.

Enzymic detection of uracil in a cloned and sequenced deoxyribonucleic acid segment

Uracil can occur in DNA either as a result of utilization of dUTP by DNA polymerases or from in situ deamination of cytosine. The latter results in transition mutations following the next round of replication. We describe a technique for the detection of uracil in DNA by a modification of the Maxam-Gilbert sequencing procedure. Reaction of end-labeled DNA with uracil-DNA glycosylase followed by 1 M piperidine results in scission products corresponding to locations of uracils. These are detected by autoradiography following electrophoresis through a sequencing gel. Comparison of these scission products with the DNA sequences elucidates the mechanism of origin of the DNA uracils. The technique was tested with a cloned human DNA sequence grown in a dut-,ung-strain of Escherichia coli, which incorporates uracil in place of thymine in its DNA, and by chemical deamination of cytosines in that sequence. The technique was expanded by use of alkaline and enzymic probes to investigate possible accumulation of uracil, base losses, and other modifications in human liver and brain DNA. No damaged DNA moieties were detected. This method is applicable to the study of any recoverable reiterated sequence by any enzyme preparation that can recognize modifications in DNA.

Differences in the distribution of O-sulphate groups of cell-surface and secreted heparan sulphate produced by human neuroblastoma cells in culture

Confluent cultures of a human neuroblastoma cell line (CHP100) were incubated for 48 h with d-[1- 3H]glucosamine and sodium [ 35S]sulphate. Radioactive glycosaminoglycans were analysed in the growth medium, rapid trypsin digest of the cell monolayer and a 1% (w/v) Triton/0.5 M NaOH extract of the final cell pellet. Sulphated glycosaminoglycans co-chromatographed when eluted by NaCL gradient from DEAE-cellulose. The medium contained mainly chondroitin sulphates, whereas the cell surface was enriched in heparan sulphate. Heparan sulphate was isolated as chondroitinase ABC-resistant material and treated with nitrous acid. Analysis of the scission products on Bio-Gel P-10 yielded fragments varying in size from single disaccharides to glycans consisting of nine disaccharide units. Cell-surface and medium heparan sulphate had respectively 52% and 54% N-sulphated glucosamine residues distributed in similar patterns along the polymer chain. The N: O-sulphate ratio of neuroblastoma heparan sulphate was 1.1:1. Analysis by high-voltage electrophoresis of di- and tetrasaccharide products produced by nitrous acid treatment showed that the distribution of ‘O’- sulphate groups differed strikingly between heparan sulphates from the medium and cell-surface compartments. A di- O-sulphated tetrasaccharide was identified in both heparan sulphate species. The absence of detectable amounts of 35[S]sulphate associated with fragments larger than tetrasaccharide supports the close topographical association of N-sulphate and O-sulphate groups.

Role of the cellular matrix in haemopoiesis. I. Synthesis of glycosaminoglycans by mouse bone marrow cell cultures.

Haemopoietically active mouse bone marrow cultures, incubated for 48 h with [3H]glucosamine and Na2(35)SO4, synthesized radiolabelled hyaluronic acid, heparan sulphate and chondroitin sulphate. Heparan sulphate was enriched in a trypsin extract of the adherent cells whereas hyaluronic acid and chondroitin sulphate were distributed mainly to the culture medium. Analysis of nitrous acid scission products of heparan sulphate by gel chromatography demonstrated the close association of N- and O-sulphate groups along the polysaccharide chain. Chondroitinase AC degradation established the copolymeric nature of chondroitin sulphate in which about 38% of the hexuronic acid residues were in the form of GlcUA. Studies on non-haemopoietic cultures, derived from W/Wv mice or from normal marrow cells maintained in foetal calf serum instead of horse serum, indicated that adherent stromal cells were the major source of glycosaminoglycans.

Synthesis of glycosaminoglycans by cloned bovine endothelial cells cultured on collagen gels.

Sulphated glycosaminoglycans have been analysed in cloned bovine aortic endothelial cells cultured on collagen gels after incubation with [3H]glucosamine and Na2(35)SO4. Radioactive products were analysed in the culture medium, in sequential collagenase and trypsin extracts of the cell monolayer and the associated extracellular matrix, and in the remaining viable cells. Heparan sulphate and chondroitin sulphate were found in each compartment: the heparan sulphate had a low degree of sulphation (approximately 0.4 N-sulphate and 0.2 O-sulphate groups per disaccharide unit on average). In the nitrous acid scission products of heparan sulphate, O-sulphated substituents were confined to disaccharide and tetrasaccharide fragments, indicating that local regions of the chain (which might be susceptible to excission by the platelet endoglycosidase) are highly sulphated. Only minor structural differences in heparan sulphate were observed between the various compartments. In contrast the chondroitin sulphate found in the collagenase extract had a higher iduronic acid content than corresponding material in the trypsin extract and the culture medium, indicating that collagenase and trypsin may extract glycosaminoglycans from different regions of the extracellular and pericellular matrix.