OCCURRENCE OF THE SINGLET-OXYGEN MECHANISM IN PHOTODYNAMIC OXIDATIONS OF GUANOSINE*

Abstract —Photosensitized oxidations of guanosine in aqueous methanol were investigated with a variety of sensitizers, and several experimental tests for the participation of singlet oxygen were examined. It has been shown that the dye-sensitized photooxidation of guanosine proceeds by both singlet-oxygen and Type I mechanisms, and that the efficiency of the singlet-oxygen mechanism is strongly dependent on photosensitizer type.     

顺铂,碳铂与鸟苷,L—蛋氨酸反应动力学比较研究

自发现顺铂具有抗癌活性以来,人们一直致力于它的抗癌机理研究。很多结果表明顺铂与蛋白质核苷酸(DNA)作用时主要与DNA中鸟嘌呤的N_7配位,通过形成链内交联作用造成DNA损伤。顺铂在显示抗癌活性的同时,也表现出较强的毒副作用,有文献认为毒性可能与形成Pt—S键抑制生物体内的巯基酶有关。     

INTERACTION BETWEEN PROFLAVINE AND GUANOSINE 5'-PHOSPHATE: IMPORTANCE OF PHOTOEXCITATION

Abstract— The stoichiometry of the complex formed between the mutagenic drug proflavine and the nucleotide guanosine 5'-phosphate has been demonstrated to be 1:1. Its association constant at room temperature is found to increase from 310 M ^-1 when proflavine is in its ground electronic state, to 1550 M ^-1, when proflavine is in its first excited singlet state. Thus, light absorbed by the drug alters its reactivity which, in turn, results in an appreciable increase in its ability to bind to the nucleotide. The enthalpy and the entropy of the ground state proflavine-guanosine 5'-phosphate complex formation are -12.6 kJ/mol (-3 kcal/mol) and -6.7 J/mol/deg (-1.6 cal/mol/deg), respectively. The implications of these findings concerning the proflavine-DNA interaction as well as the possible importance of electronic excitaton in acridine mutagenesis are discussed. An appraisal of the methodology for analyzing fluorescence quenching data is also given.     

EFFECTS OF ULTRAVIOLET RADIATION ON THE MITOTIC CYCLE AND DNA, RNA AND PROTEIN SYNTHESIS IN MAMMALIAN EPIDERMIS IN VIVO

Abstract— Acute effects of ultraviolet radiation on the mitotic cycle and macromolecular synthesis were investigated on hairless mouse epidermis in vivo. Colcemid was used to arrest mitoses in metaphase and thus allow more accurate mitotic counts. The radioactive tracers, TdR-³H, cytidine-³H, and the amino acids, histidine-³H and methionine-³H were used to examine DNA, RNA and protein synthesis, respectively. Using these techniques, we found that wavelengths shorter than 320 nm markedly inhibited mitosis, increased the basal cell turnover time and depressed DNA, RNA and protein synthesis within the first few hours post-irradiation. By 24hr, recovery and acceleration of these functions were in progress, reaching a peak by 48–72 hr and persisting though to a lesser degree for 7 days. This stage of acceleration was associated with epidermal hyperplasia and most likely represented post-injury cell renewal.     

THE ACTION MODE OF CISPLATIN ON DNA-SYNTHESIS AND CHARACTERIZATION OF N7 N1 CROSS-LINKAGE MODEL COMPOUND OF PLATINUM AND GUANOSINE

The title Pt-Guo compound was synthesized and isolated by the reaction between cis-[Pt(NH3)2(H2O)2](NO3)2 and guanosine of mol ratio1:1 in a neutral aqueous solution followed by careful purification with partial crystalization and recrystallizatlon from water at 0℃.The compound was characterized by elemental analysis,molecular weight determination,DTA,and C NMR spectroscopy and its formulawas found to be [8(GuoH-1)7 (OH)2] (NO3)7 8H2O.The isolation of the compound provides direct evidence for the intrastrand cross-linkage between cisplatin and DNA through N7,N1 atoms of two adjacent guanines.     

ELECTRONICALLY EXCITED SPECIES IN THE PEROXIDASE CATALYZED OXIDATION OF INDOLEACETIC ACID. EFFECT UPON DNA AND RNA

Abstract— The electronically excited species generated in the peroxidase (oxidase) catalyzed oxidation of the plant hormone indole-3-acetic acid is an excited state of indole-3-carboxaldehyde.
The chemiexcited species is able to induce in DNA the same alterations as observed with light or with enzyme-generated triplet acetone. The chemiexcited species can also alter r-RNA.     

IDENTIFICATION AND LOCALIZATION OF PHOTOALKYLATED BASES IN DNA FRAGMENTS

Abstract— Antibodies directed toward 8-(2-hydroxy-2-propyI)-deoxyguanosine-5'-monophosphate (8-hpdGMP), a product of the photoalkylation of deoxyguanosine-5'-monophosphate with 2-propanol, were shown to be highly specific and sensitive in detecting 8-hpdGMP residues in photoalkylated DNA.
The antibodies were further used to determine the distribution of modified guanine residues in DNA. The irradiated DNA was digested with restriction enzymes and the fragments obtained were separated electrophoretically and blotted onto diazotized paper. The covalently bound DNA fragments were probed with the antibodies and then with ¹²⁵I Staphylococcus aureus protein A. These experiments indicate a non-random distribution of modified guanine residues in øX174 RF DNA molecules.     

EXCISION OF CYCLOBUTANE DIMERS IN GENOMIC AND EPISOMAL DNA IN HUMAN CELLS

Abstract Direct determination has been made of cyclobutyl pyrimidine dimer induction and excision repair in an episomal SV40 DNA population in vivo . Maintaining SV40-transformed human (GM637) cells in confluent culture results in amplification of a mutant SV40 episome to high copy number. T4 endonuclease V was used to quantify the induction and repair of cyclobutane dimers in the SV40 episome and genomic DNA of the same cells. Differences in both parameters were observed cyclobutane dimers were induced at 1.5–2-fold greater frequency in episomal DNA and excised at a reduced rate compared to genomic DNA in the host cells.     

ULTRASTRUCTURE OF THE NUCLEOLUS AND REPLICATION SITES OF RIBOSOMAL DNA IN Allium cepa

Our conventional EM observations indicate that the nucleolus of Allium cepa is composedof the fibrillar centre (FC), fibrillar component (F) and granular region (G). FC is an elec-tron-lucid zone which contains condensed or loosened chromatin. F is a circular, highly elec-tron-dense component which surrounds FC and consists of compact fibrils. EM autoradiographic(EM ARG) studies with ~3H- UdR reveal that the synthesis of ribosomal RNA (rRNA) takesplace in F. G is situated around F and composed of granules about 25 to 30 nm in diameter.EM ARG studies with ~3H- TdR demonstrate that silver grains are predominantly located in Gof the nucleolar periphery and the region of the nucleolus-associated chromatin. In addition,~3H- TdR labels are also present, but with a much lower frequency, in FC. F and G of the nu-cleolar interior. According to the distribution and sphere of silver grains in different compo-nents of the nucleolus, we suggest that the replication sites of ribosomal DNA (rDNA) aremainly located in     

8-METHOXYPSORALEN DNA INTERSTRAND CROSS-LINKING OF THE RIBOSOMAL RNA GENES IN Tetrahymena thermophila. DISTRIBUTION, REPAIR AND EFFECT ON rRNA SYNTHESIS

Abstract— The distribution and repair of 8-methoxypsoralen-DNA interstrand cross-links in the ribosomal RNA gencs (rDNA) in Tetrahymena thermophila have been studied in vivo by Southern blot analysis. It is found that the cross-links at a density of ≤ 1/2 × 10⁴ base pairs (bp) are distributed equally between three domains (terminal spacer, transcribed region and central spacer) as defined by restriction enzyme analysis ( Bam HI and Cla I). It is furthermore shown that a dosage resulting in approximately one cross-link per rDNA molecule (21 kbp, two genes) is suficient to block KNA synthesis. Finally, it is shown that the cross-links in the rDNA molecules are repaired at equal rate in all three domains within 24 h and that RNA synthesis is partly restored during this repair period. The majority of the cells also go through one to two cell divisions in this period but do not survive.     

INHIBITION OF DNA REPAIR AND PRODUCTION OF SINGLE-STRAND BREAKS IN ESCHERICHIA COLI BY REDUCTONE

Abstract— Reductone (HOCH₂COCHO), a keto-aldehyde produced by thermal degradation of some sugars, at alkaline pHs, blocks the excision repair of DNA lesions in uv-irradiated wild type Escherichia coli. This probably occurs as a result of inhibition of the exonucleolytic activity of DNA polymerase I. In addition, reductone alone induces DNA single-strand breaks. Repair of this damage is mainly dependent on the polA gene products.     

QUANTITATION OF DNA DAMAGE IN NON-RADIOACTIVE DNA

Three principal methods have been developed for measuring femtomoles of damage in nanogram quantities of non-radioactive DNA. Lesions which can be quantified include single and double strand breaks, alkali labile sites including apurinic and apyrimidinic sites, and pyrimidine dimers. The first in vitro method measures the conversion of supercoiled DNA to relaxed or linear molecules, and can detect up to four lesions per molecule. The second in vitro method (supercoil depletion) assesses the fraction of intact linear molecules of homogeneous length, and allows detection of 8 lesions/molecule. The third method, measurement of molecular length distributions of DNAs of heterogeneous length, reveals the extent of DNA damage and repair in vivo or in vitro.     

PHOTOLYSIS OF IODODEOXYCYTIDINE IN DNA AND RELATED POLYNUCLEOTIDES: WAVELENGTH DEPENDENCE AND ENERGY TRANSFER*

Abstract— 5'-Iodocytosine (IC) containing denatured DNA and poly(C) were prepared and the photoinduced loss of iodine measured for irradiation at wavelengths between 240 and 313 nm. The following intrinsic quantum yields (Φ_INT) were obtained for irradiation at λ_ex > 300 nm where only IC absorbs: denatured DNA (0.01), poly(C) (0.013), apurinic acid (0.018) and IdCMP (0.026). These results suggest that geometrical or structural restraints in the polymer, which increase with the degree of base stacking, inhibit the loss of iodine from an excited IC residue. The variation in the photochemical cross section for iodine photolysis was measured as a function of the wavelength of irradiation and found to vary in a manner which indicates that absorption by noniodinated residues can lead to iodine photolysis. It is proposed that energy transfer from neighboring bases to an adjacent IC residue takes place, resulting in an action spectrum for iodine photolysis which reflects absorption of excitation energy by noniodinated as well as iodinated residues. The contribution due to energy transfer in denatured DNA was estimated to be from not more than a single base located on either side of an IC residue. The degree of transfer was slightly less in iodinated poly(C) and decreased 4-fold following depurination of the DNA. These results are consistent with a structurally dependent energy transfer process in which IC, because of its lower lying singlet state, can act as an energy trap.     

IDENTIFICATION OF PARTIAL MUTATIONAL SITES IN HUMAN MITOCHONDRIAL DNA IN THE CHINESE AND ITS SIGNIFICANCE

A simple method for the identification of mutational sites in human mitochondrial DNA (mtDNA) was described. It was based on the human Cambridge sequence as a relative standard sequence and a single base pair substitution in mtDNA as a unique mutational form. The partial mutational sites can be determined using this method which was characterized by combining the restriction mapping with the analysis for the table of human mtDNA potential mutational sites with rapidity and simplicity. In the meanwhile, six mtDNA mutational sites found in Chinese population were identified by means of this method.     

INDUCTION OF FREE RADICALS IN DNA BY PROFLAVINE AND VISIBLE LIGHT: INFLUENCE OF OXYGEN AND IONIC STRENGTH

Abstract —The photosensitization of native DNA is observed as an induction of free radicals in the DNA moiety of proflavine-DNA complexes. The intensity of the electron paramagnetic resonance spectra (at 77 K) is a measure of the number of free radicals present in frozen solutions of DNA-proflavine complexes after irradiation with visible light (Λ > 320 nm). In the absence of O₂, the photosensitization is significant but very low; it increases slightly with increasing NaCl ionic strength; it appears to be due to intercalated dye molecules and the qualitative analysis of the spectra obtained shows that mainly thymidine is involved. The reaction measured after saturation with O₂ is the same as the reaction in air but is quantitatively higher; the free radicals observed are peroxides. This induction of free radicals appears to be due to the intercalated dye molecules, each molecule acting independently. The important observation is a very sharp and large (around a hundred-fold) increase in the photosensitizing efficiency of the bound dye molecules occurring in NaCl between μ, # 0–25 and μ= 0–5 and in MgCl₂ between μ# 0–01 and μ=0–1.     

OCCURRENCE OF PYRIMIDINE-RICH TRACTS IN ASCITES TUMOR DNA AND THE FORMATION OF UV-INDUCED THYMINE DIMERS

Abstract— Analysis of the distribution of pyrimidine-rich tracts (up to decanucleotides) in ascites tumor DNA revealed that these tracts occur predominantly in repetitive sequence of DNA. UV irradiation of ascites DNA resulted in preferential formation of thymine dimers in the pyrimidine-rich tracts as compared to other regions of DNA.     

PHOTOALKYLATION OF PURINES IN DNA

Abstract— Ultraviolet light irradiation of DNA in the presence of alcohols such as 2-propanol leads to hydroxyalkylation of the purine bases. The purine photoproducts were identified as C-8 hydroxyalkyl derivatives of adenine and guanine by chromatographic mobility, co-crystallization with authentic samples, and by photoreversal to the original purines. Experiments are reported which demonstrate that the photoalkylation reaction is dependent upon secondary structural alterations in DNA. It is suggested that the photoalkylation of purines proceeds through free-radical intermediates.
The presence of alcohols during irradiation considerably decreases pyrimidine-dimer formation.     

CHANGE IN THE BASE COMPOSITION OF MESSENGER RNA OF ESCHERICHIA COLI BY ULTRAVIOLET IRRADIATION AND ITS RECOVERY

Abstract— The base composition of messenger RNA in Escherichia coli B/r and B _8–1 irradiated with ultraviolet (u.v.) light has been examined. The experimental results are as follows: (1) the synthesis of rapidly labeled RNA does not stop in ultraviolet irradiated bacteria. (2) The rapidly labeled RNA in irradiated cells shows a change in base composition corresponding to the formation of pyrimidine dimers in DNA molecules. The mole per cent of adenine component is increased with ultraviolet dose. The ratio of purine/pyrimidine becomes larger and the GC content smaller. (3) The base composition of the rapidly labeled RNA in irradiated bacteria reversed to that in unirradiated cells, when the irradiated cells were reactivated by experimental procedures for photoreactivation or dark reactivation. The reversion in the base composition corresponds well to the decrease in the amount of thymine dimers in DNA molecules. (4) The mechanism of the change in the base composition of rapidly labeled RNA caused by ultraviolet irradiation is discussed.