REPAIR OF 8-METHOXYPSORALEN INDUCED DNA INTERSTRAND CROSS-LINKS IN Tetrahymena thermophila

Abstract— The protozoan Tetrahymena thermophila was treated with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation and the DNA-repair response was studied. Following the treatment a lag-period in cell proliferation was observed, the duration of which was proportional to the amount of psoralen used, and both swelling and deformation of the cells were observed. The treatment with 3 μg 8-methoxypsoralen/mℓ and a light dose of 8 kJ m^-2 resulted in a 10-fold decrease in DNA and RNA synthesis activity, while the protein synthesis was only moderately affected. Using the same conditions the lag-period was 30 h, and during this time the psoralen induced DNA interstrand cross-links were removed. Alkaline elution experiments showed that the repair process involves DNA single strand scissions, whereas no double strand DNA scissions were detected.     

PHOTOSENSITIZING EFFECTS OF 8-METHOXYPSORALEN ON THE SKIN OF HAIRLESS MICE—I. FORMATION OF INTERSTRAND CROSS-LINKS IN EPIDERMAL DNA

Abstract— The photomediated induction of interstrand cross-links by 8-methoxypsoralen has been measured in the epidermal DNA of hairless mice. Equivalent efficiencies for cross-link induction were determined for HRS/J/Anl and SKH: hairless-1 mice. A wavelength dependence on the relative efficiency of cross-link induction was observed; a broad spectrum light source, 300–400 nm, was approximately 5 times more effective in cross-link formation than a 365 nm light source. Repeated exposure to 8-methoxypsoralen followed by ultraviolet light, 5 times a week for 6 weeks, altered epidermal thickness and resulted in a decreased efficiency for DNA cross-link formation.     

FACTORS INFLUENCING THE NEAR-UV EFFECT ON DNA IN THE PRESENCE OF 8-METHOXYPSORALEN: RENATURABILITY OF DNA.

Factors affecting the near-UV induced reaction of calf thymus DNA in the presence of 8-methoxypsoralen were examined. DNA became more renaturable with increasing concentration of 8-methoxypsoralen and fluence. Existence of paramagnetic ions, Mn²⁺, Co²⁺, or Ni²⁺, largely repressed the photoreaction. It was ascertained that the highest renaturability was attained by the illumination with light between 310 and 345 nm.     

THIOPYRONINE- AND 8-METHOXYPSORALEN-SENSITIZED PHOTODYNAMIC EFFECT ON CELL GROWTH, COLONY FORMING ABILITY and RNA SYNTHESIS IN Saccharomyces MUTANTS DEFICIENT IN DNA REPAIR

Abstract –We compared the photodynamic effects of thiopyronine (TP) and visible light, and 8-methoxypsoralen (8-MOP) and ultraviolet A (UV-A) light, on growth, colony forming ability and RNA synthesis in a repair-proficient Saccharomyces strain and three mutants deficient in DNA repair mechanisms (DNA repair assays). With 8-MOP and UV-A repair-deficient mutants were significantly more sensitive than the repair-proficient strain indicating that the system is sensitive for the detection of DNA damage. With TP and visible light, the photodynamic effects were comparable in the mutants and the control, indicating no DNA damage. These results support previous work showing that the main target of TP photosensitization in eukaryotes is not nuclear DNA.     

PHOTOTOXIC EFFECTS OF FOUR PSORALENS ON L1210 CELLS. THE CORRELATION WITH DNA INTERSTRAND CROSS-LINKING

L1210 mouse leukaemia cells were treated with psoralen [S-methoxy-(XMOP), 4,5′,8-trimethyI-(TMP), 4′-hydroxymethyl-4,5′,8-trimethyl-(HMT) or 4′-amino- methyl-4,5′,8-trimethylpsoralen (AMT)] in combination with long wavelength ultraviolct irradiation (Λ～ 365 nm). In order to investigate the relative photobiological activities of the psoralens, cell viability and DNA-synthesis activity as well as psoralen-DNA photoaddition and DNA interstrand cross-linking were measured after the treatment. In all assays the activity ranking order was found to he: TMP > HMT > AM7 > 8MOP. Furthermore, a direct correlation between phototoxicity, psoralen induced DNA interstrand cross-links and inhibition of DNA synthesis was indicated. Finally, psoralen uptake by the cells appears to be an important determinant for phototoxicity, whereas their DNA photoreactivity does not.     

RESEARCH NOTE: THIOPYRONINE-AND 8-METHOXYPSORALEN-SENSITIZED PHOTODYNAMIC EFFECT ON CELL GROWTH, COLONY FORMING ABILITY AND RNA SYNTHESIS IN Saccharomyces MUTANTS DEFICIENT IN DNA REPAIR

Abstract: We compared the photodynamic effects of thiopyronine (TP) and visible light, and 8-methoxypsoralen (8-MOP) and ultraviolet A (UV-A) light, on growth, colony forming ability and RNA synthesis in a repair-proficient Saccharomyces strain and three mutants deficient in DNA repair mechanisms (DNA repair assays). With 8-MOP and UV-A repair-deficient mutants were significantly more sensitive than the repair-proficient strain indicating that the system is sensitive for the detection of DNA damage. With TP and visible light, the photodynamic effects were comparable in the mutants and the control, indicating no DNA damage. These results support previous work showing that the main target of TP photosensitization in eukaryotes is not nuclear DNA.     

THE EFFECT OF DARK COMPLEXING ON THE PHOTOSENSITIZED FORMATION OF 8-METHOXYPSORALEN CROSS-LINKS WITH DNA*

The near-UV dose at 365 nm required for the formation of 8-methoxypsoralen (8-MOP) cross-links with calf thymus DNA was found to be insensitive to the concentration ratio of nucleotides to 8-MOP over the range 100:1 to 5:1. The proposed explanation is that only the dark-complexed 8-MOP contributes to the photosensitized formation of monoadducts and cross-links. A kinetics analysis indicates that the fluence required for cross-linking should be inversely proportional to the square root of the fraction of dark-complexed 8-MOP per nucleotide (the binding ratio), a quantity that is insensitive to the ratio of nucleotides to 8-MOP. These findings predict a small dependence of photosensitivity on the total 8-MOP concentration in cellular systems in which cross-links are the major lethal lesion.     

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.     

DNA TURNOVER IN BUFFER-HELD ESCHERICHIA COLI AND ITS EFFECT ON REPAIR OF UV DAMAGE

Abstract— Continuous DNA degradation and resynthesis, without a net change in cellular DNA content, were observed in buffer-held, non-irradiated E. coli B/r. This constant DNA turnover probably involves most of the genome and reflects random sites of DNA repair due to the polA-dependent excision-resynthesis repair pathway. Under these non-growth conditions, it appears that at any given time there is a minimum of one repair site per 6.5 × 10⁶ daltons DNA, each of which is at least 160 nucleotides long.
While the amount of DNA degradation is not influenced by prior exposure to UV radiation, the synthetic activity decreases with increasing UV fluence. We suggest that when sites of DNA turnover occur opposite to cyclobutyl dipyrimidines in UV-irradiated cells, repair of the latter damage can be prevented. This implies that both beneficial and deleterious processes take place in irradiated buffer-held cells, and that cell survival depends on the delicate balance between DNA turnover and repair of UV-damage. Based on these findings, we propose a model to explain the limited repair observed during post-irradiation liquid-holding and to account for the large difference in cell survival between irradiation at low fluence rates (fluence-rate dependent recovery) and at high fluence rates followed by liquid-holding (liquid-holding recovery).     

THE INTERACTION OF MELANIN WITH 8-METHOXYPSORALEN: EFFECT ON RADIATIVE AND NONRADIATTVE TRANSITIONS. A FLUORESCENCE AND PULSED PHOTOACOUSTIC STUDY

Abstract: The interaction of 8-methoxypsoralen (8-MOP) with synthetic eumelanin was investigated using static and time-resolved fluorescence and pulsed photoacoustic calorimetry. Due to the strong overlap of the absorption bands of melanin and 8-MOP, a method is presented to account for the systematic errors introduced by the optical filter effect exerted by each absorbing species in the fluorescence and the photoacoustic measurements. As a preliminary step to the understanding of the nonradiative behavior of the psoralen-melanin complexes, the photoacoustic parameters of 8-MOP in various solvents were determined. Spectroscopic data indicate the absence of interaction at the ground-state level, whereas the singlet excited state of 8-MOP is quenched by the pigment; the average fluorescence lifetimes are independent of the melanin concentration, thus indicating a static quenching mechanism. The photoacoustic data show that the quenching process involves an increased intersystem crossing probability, which is almost unaffected by the presence of oxygen, as expected for a molecule essentially acting as a type I photosensitizing agent.     

EFFECTS OF 8-METHOXYPSORALEN AND NEAR ULTRAVIOLET RADIATION ON THE SURVIVAL OF THE LOWER EUKARYOTE D. Discoideum

Abstract— Seven axenic wild-type and repair-deficient mutant strains of the cellular slime mold Dictyostelium discoideum have been treated with the furocoumarin 8-methoxypsoralen (8-MOP) up to 50 μg/mζ and then exposed to near ultraviolet light (UVA 320-400 nm) up to 21 kJ/m². Fluence-response survival curves exhibit shoulders at lower fluences and an exponential lethal response at higher fluences. Neither the psoralen alone nor the irradiation alone produced any measurable lethal effect. Wild-type strains, which show resistance to 254 nm UV and gamma radiation, also show resistance to psoralen plus UVA. The moderate sensitivity of a rad D repair-deficient mutant strain and the extreme sensitivity of a rad B mutant strain to 8-MOP plus UVA parallel their responses to UV and gamma radiation. However a rad C mutant which is sensitive to UV, exhibits wild-type response to photoactivated psoralen.     

DIFFERENT EFFECTS OF RAD GENES OF SACCHAROMYCES CEREVISIAE ON INCISIONS OF INTERSTRAND CROSSLINKS AND MONOADDUCTS IN DNA INDUCED BY PSORALEN PLUS NEAR UV LIGHT TREATMENT

Abstract— The RAD1, RAD2, RAD3 and RAD4 genes of Saccharomyces cerevisiae are required for incising DNA containing UV induced pyrimidine dimers or psoralen plus 360 nm light induced interstrand crosslinks. We have now determined if these genes are also required for incising DNA at psoralen plus 360 nm light induced monoadducts. For distinguishing between incision breaks occurring at crosslinks and at monoadducts. we have used the cdc9-2 mutant, defective in DNA ligase activity at the restrictive temperature, and the radl-2 cdc9-2, rad2-5 cdc9-2 , rad3-2 cdc9-2 and rad4-4 cdc9-2 double mutant combinations. We conclude that the radl, rad2 , and rad4 mutants are defective in incising DNA both at crosslinks and monoadducts, whereas the rad3 mutant is proficient in incising DNA at monoadducts but not at crosslinks.     

TOXICITY, DNA DAMAGE AND INHIBITION OF DNA REPAIR SYNTHESIS IN HUMAN MELANOMA CELLS BY CONCENTRATED SUNLIGHT

A 1 m diameter water lens was used to focus solar radiation, giving an 8-fold concentration of the total spectrum and a cytocidal flux similar to that of laboratory UV sources. Survival curves for human melanoma cells were similar for sunlight and 254 nm UV, in that D _q, was usually larger than D _o. An xeroderma pigmentosum lymphoblastoid line was equally sensitive to both agents and human cell lines sensitive to ionizing radiation (lymphoblastoid lines), crosslinking agents or monofunctional alkylating agents (melanoma lines) had the same 254 nm UV and solar survival responses as appropriate control lines. Two melanoma sublines derived separately by 16 cycles of treatment with sunlight or 254 nm UV were crossresistant to both agents. In one melanoma cell line used for further studies, DNA strand breaks and DNA-protein crosslinking were induced in melanoma cells by sunlight but pyrimidine dimers (paper chromatography) and DNA interstrand crosslinking (alkaline elution) could not be detected. The solar fiuence response of DNA repair synthesis was much less than that from equitoxic 254 nm UV, reaching a maximum near the D _o value and then declining; semiconservative DNA synthesis on the other hand remained high. These effects were not due to changes in thymidine pool sizes. Solar exposure did not have a major effect on 254 nm UV-induced repair synthesis.     

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     

COMPARATIVE STUDIES ON THE PHOTOSENSITIZING POTENCY OF 5-METHOXYPSORALEN and 8-METHOXYPSORALEN AS MEASURED BY CYTOLYSIS IN PARAMECIUM CAUDATUM AND TETRAHYMENA PYRIFORMIS, AND GROWTH INHIBITION AND SURVIVAL IN CANDIDA ALBICANS

Abstract— The photosensitizing potencies of 5-methoxypsoraIen and 8-methoxypsoralen were investigated using the following biological end-points for lethality: (i) cytolysis in the protozoans Paramecium caudatum and Tetrahymena pyriformis, (ii) inhibition of growth in the yeast Candida albicans and (iii) survival as measured by colony counts in Candida albicans. In all cases, 5-methoxypsoralen proved to be the more potent photosensitizing agent. The preliminary action spectra of the 2 compounds for growth inhibition in C. albicans were similar and showed maximal spectral efficiency in the 320–340 nm waveband. The molecular basis for the described end-points is unknown. Although it is well known that 5-methoxypsoralen and 8-methoxypsoralen readily photoreact with DNA, it is considered that photo-reaction with protein should also be given serious consideration as the possible lethal event. The superior effect of 5-methoxypsoralen is in accordance with some of the physical and photochemical properties of this molecule, but this result is at variance with other studies in different test systems that have been used to compare the photobiological efficacy of these 2 compounds.     

DSC测试中样品内部的温度分布及其对比热测试的影响

由傅里叶导热定律结合Tools-Narayanaswamy-Moynihan(TNM)松弛模型提出了一种计算聚合物样品内温度分布的方法.对PS的计算结果表明温度梯度随样品厚度、升温速率增大而增大,对于1.0 mm的PS以1 K/min降温后再以10 K/min升温时样品内部最大温差为0.882 K.在此基础上提出了不同样品厚度、不同热处理条件下聚合物测定比热的计算方法,结果表明计算结果与实测数据基本一致.     

BLUE-LIGHT INDUCED DEVELOPMENT OF CHLOROPLASTS IN ISOLATED SEEDLING ROOTS. PREFERENTIAL SYNTHESIS OF CHLOROPLAST RIBOSOMAL RNA SPECIES

Excised roots of pea seedlings (Pisum sativum var. “Alaska”) cultured in a synthetic medium under sterile conditions exhibit differentiation of functional chloroplasts from leucoplasts when irradiated with blue light (350–550 nm). This transition is a relatively slow process; nevertheless, the chloroplasts formed in blue light compare very well to leaf chloroplasts as far as microstructure and photosyn-thetic activities are concerned. Apparently certain activities of the apical meristem are mandatory in bringing about a transition from leucoplasts to chloroplasts in blue light. After short-time labelling with [^jH]uridine the synthesis of plastid ribosomal RNA (rRNA) was studied either during irradiation with blue and red light (600–700 nm), respectively, or in darkness. Polyacrylamide gel electrophoresis revealed that in blue light the synthesis of specific chloroplast rRNA species with molecular weights of 1.1 × 10⁶ and 0.56 × 10⁶ daltons is markedly stimulated. In contrast, in dark cultured roots these RNA species were synthesized to a limited extent only whereas the cytoplasmic rRNA species of 1.3 × 10⁶ and 0.7 × 10⁶ daltons molecular weight were preferentially formed. The same holds true for roots irradiated with red light.     

溶剂对聚乙炔合成及性能的影响

聚乙快的不稳定性及难以加工影响了其应用.最近,Naarmann通过陈化在硅油中的Ti(OBu)_4~-AlEt_3催化体系及加入不同量的Li-(n-Bu)制得了拉伸后电导率达10~5S/cm的聚乙炔膜,此膜的电导和形态在空气中几个月几乎不发生改变.我们曾报导用加热的甲苯-Ti(OBu)_4~--AlEt_3催化体系合成高性能聚乙炔膜.本文进一步讨论不同溶剂对高性能聚乙炔(HPPA)合成及性能的影响.