Continuous-flow system for large-scale ultraviolet irradiation of bacterial cells.

A quartz-flow-cell system for irradiation of large volumes of Escherichia coli cultures with ultraviolet light is described. With this system kilogram quantities of irradiated cells can be obtained for biochemical studies. Changes in respiration and in specific activities of superoxide dismutase and catalase, after an ultraviolet treatment that reduced viability of culture samples to 0.2%, were in good agreement with those for cultures irradiated (52J/m2) by a conventional small-scale method to produce the same reduction in viability.     

Delayed ultraviolet light-induced cessation of respiration by inadequate aeration of Escherichia coli.

Inadequately aerated Escherichia coli B/r cultures did not shut their respiration off 60 min after ultraviolet light (52 M/m2 at 254 nm) as they did when well supplied with oxygen. Since cessation of respiaration is associated with cell death, the result suggested that oxygen toxicity by superoxide radicals generated by cell metabolism might be responsible for cell death. The specific activity of superoxide dismutase, which scavenges O2- radicals, increased twofold after 90 min of adequate aeration, but the specific activity of catalase remained constant. Respiration and viability of irradiated cells were affected not at all by the presence of superoxide dismutase and only slightly by the presence of catalase. Metal ions such as Mn2+ and Fe2+ inducers of superoxide dismutase, had no effect on respiration and viability. When irradiated cells were incubated under N2 for 90 min, the respiration, growth, and viability time-course responses were the same as for the cells not exposed to anareobiosis. We conclude that superoxide anions generated at the time of irradiation play no part in cessation delays the ultraviolet light-induced synthesis of proteins responsible for the irreversible cessation of respiration.     

Ultraviolet-induced changes in the infectivity of Agrobacterium tumefaciens

The infectivity of Agrobacterium tumefaciens strain B6 irradiated with short-wavelength ultraviolet light was followed as a function of dose. Previously reported enhancements of B6 infectivity by ultraviolet irradiation, in samples inoculated after 1.75 hr of dark incubation at 27 C, or immediately following irradiation, were found to occur most frequently after losses in cell viability of 60% and of 90% or more, respectively. Changes in colony-forming ability and tumor-initiating ability with increasing dose showed no obvious correlation until the maximal infectivity promotion of samples inoculated immediately after irradiation was reached. Thereafter, both bacterial responses typically decreased in parallel. With low dose rates, infectivity promotions were obtained with less than 10% loss in cell viability. Data for tumor appearance and tumor growth resulting from inoculations with irradiated cultures showed no significant differences from controls, nor did the age of the bacterial culture or age of the host plant influence the response. The infectivity promotion appears to result from an increase in the proportion of viable cells that will subsequently initiate tumors. The characteristics of this ultraviolet infectivity promotion are shown to be most similar to those found in prophage and bacteriocin induction.     

Ultraviolet reactivation of lambda phage: assay of infectivity of DNA molecules by spheroplast transfection

Prior irradiation of non-lysogenic bacteria by ultraviolet light leads to an increase in the viability of infecting irradiated λ phage (ultraviolet reactivation). Similarly, u.v. irradiation of wild type or uvrD bacteria lysogenic for λcIind^? increased the fraction of closed circular duplex phage DNA molecules formed after infection with u.v.-irradiated λ phage. The closed circular molecules isolated from the irradiated lysogens were shown to be free from u.v. damage by a spheroplast transfection assay. The increase of closed circular molecules is sufficient to explain the ultraviolet reactivation observed by the increase of viability of irradiated phage.In ultraviolet reactivation, damage must be erased on irradiated DNA molecules and the repair is independent of total replication of phage genomes, exchange of sister chromatids or recombination between phage genomes. Protein synthesis is necessary to increase the level of closed circular molecules of irradiated λ phage after irradiation of bacteria.     

Slow clones,reduced clonogenicity,and intraclonal recovery in X-irradiated L5178Y-S cell cultures

Profound, long-lasting growth disturbances and reduced viability and clonogenicity were observed in suspension cultures of L5178Y-S (LY-S) murine leukemic lymphoblasts exposed to 0.25–6 Gy of X rays. In most cases, uncloned cultures grew at a reduced rate for periods corresponding to at least 100 cell generations, even when viability of such cultures returned to the normal level. These disturbances were analyzed in clones isolated using agar-supplemented medium. A slow phenotype was much more frequent among surviving clones isolated from LY-S cell cultures irradiated with 3 Gy of X rays than among clones isolated from nonirradiated controls. Growth of individual LY-S clones was affected to different extents, regardless of the clone's viability. The slowest clones had doubling time twice as long (22 h) as that of the control (10–12 h). More than 100 slow clones isolated from irradiated and nonirradiated cultures were followed for prolonged times, and some of them were further subcloned. The slow clones showed a high degree of heterogeneity, and selection for the slowest clone produced clones with increasing proliferative impairment and decreasing cloning efficiency. These results showed that progeny of X-irradiated LY-S cells contained many slowly growing cells, and that their presence affected the growth rate for scores of cell generations. The prolonged impairment of growth rate, viability, and clonogenicity appeared to depend on heritable lesions that were overcome as a result of intraclonal recovery. All slow clones were capable of such recovery, which for clones derived from irradiated cultures typically required periods corresponding to several scores of, but in some cases > 200, cell generations. Intraclonal recovery was much more rapid in slow clones isolated from nonirradiated cultures. This finding indicated that either slow phenotype depended on different cellular changes in the two groups of clones or mechanisms of intraclonal recovery were affected by radiation.     

An attempt to stimulate mitosis in Saccharomyces cerevisiae with the ultraviolet luminescence from exponential phase cultures of this yeast

Neither cell division nor growth of Saccharomyces cerevisiae were stimulated by the ultraviolet luminescence produced by adjacent exponential phase cultures of the yeast. The study included experiments in which the inocula (density = 5 X 10(7) cells cm-3) were irradiated and in which lag phase cultures (densities = 1 X 10(6) or 5 X 10(6) cells cm-3) were irradiated for 30 min with the yeast luminescence. These results do not support the claims of earlier workers that dividing cells can stimulate mitosis in optically coupled cultures by the so-called "mitogenetic effect."     

A simple and rapid method for evaluating the survival of xeroderma pigmentosum lymphoid lines after irradiation with ultraviolet light

A simple, rapid, and reproducible test has been developed to measure the viability of cells after irradiation with ultraviolet light (UV). Epstein-Barr virus-transformed lymphoid lines, derived from patients with xeroderma pigmentosum (XP), were irradiated with UV, and the post-UV viability of the lymphoid lines was determined by the trypan blue dye exclusion method. The relative post-UV survival of the patients' lymphoid lines was similar to the relative post-UV survival of the patients' fibroblast strains.     

A simple and rapid method for evaluating the survival of xeroderma pigmentosum lymphoid lines after irradiation with ultraviolet light

Summary A simple, rapid and reproducible test has been developed to measure the viability of cells after irradiation with ultraviolet light (UV). Epstein-Barr virus-transformed lymphoid lines, derived from patients with xeroderma pigmentosum (XP), were irradiated with UV, and the post-UV viability of the lymphoid lines was determined by the trypan blue dye exclusion method. The relative post-UV survival of the patients' lymphoid lines was similar to the relative post-UV survival of the patients' fibroblast strains.     

Enhancement of monoclonal antibody production in CHO cells by exposure to He–Ne laser radiation

This study tested the effectiveness of laser biostimulation in small-scale cultures in vitro. We investigated the response of recombinant CHO cells, which are used for the production of monoclonal antibody, to low level laser radiation. The cells were irradiated using a 632.8 nm He–Ne laser in a continuous wave mode at different energy doses. We incubated the irradiated cells in small batch cultures and assessed their proliferation and productivity at various time intervals. Compared to untreated cells, the irradiated cells showed a significant increase in antibody production. Moreover, the results showed that laser irradiation did not affect viability and slightly enhanced proliferation rate.     

Effect of x-irradiation of one partner on hybrid frequency in fusions between Chinese hamster cells

Sublines derived from V79 and B150 Chinese hamster cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and thymidine kinase (TK) respectively. When these strains are cultivated together without virus treatment in HAT medium, hybrid colonies with normal enzymic patterns arise in high frequency. The ratio of proliferating hybrid colonies to the minority cell inoculum in mixed cultures is approximately 1/1000. This observation has been used to examine the effect of x-irradiation pretreatment of one cell type on the frequency of hybrid formation in mixed cultures. B150 cells (TK-) were irradiated in monolayer cultures at dose levels of 200–4000r (180 KV, 15 ma). After trypsinization, the irradiated cells were mixed with unirradiated V79 cells (subline 129, HGPRT-) and cultivated in HAT medium. The per cent decline in hybrid frequency as a function of x-irradiation was compared to the relative decline in viability of x-irradiated B150 cells when assayed in cultures alone. These curves diverged markedly, with the frequency of proliferating hybrids decreasing much less rapidly with radiation dose than survival of B150 cells. By contrast, when B150 cells were subjected to acute heat shock for graded intervals and subsequently mixed with untreated V79–129 cells, both viability of B150 cells and hybrid formation declined at the same rate. Our results suggest that lethally irradiated cells can still fuse with normal cells, and may contribute functional chromosomes to the hybrid karyotype. At limiting doses of x-irradiation this technique may be useful as a means for induced segregation of marker chromosomes.     

Enhanced Production of Virus-Inhibiting Factor (Interferon) in Human Diploid Cells by Ultraviolet Irradiation and Temperature Shift-Down after Stimulation with Newcastle Disease Virus

The production of the virus-inhibiting factor or interferon (IF) was highest in cells incubated at 37 C after inoculation with Newcastle disease (ND) virus and decreased as the incubation temperature was lowered. Shift-down of incubation temperature to 32 C or 34 C after incubation at 37 C for 4–7 hr enhanced IF production in cell cultures stimulated with ND virus, as compared with cultures incubated continuously at 37 C. Shift-down to 32 C after incubation at 37 C for 6 hr. was optimal for this enhancement of IF yield. Enhanced IF production was also observed in cell cultures irradiated by ultraviolet light 4–7 hr after stimulation with ND virus.     

Repair-induced Changes in Yeast Radiosensitivity

Potentially lethal X-ray or ultraviolet damage in the diploid yeast, Saccharomyces cerevisiae, can be reversed if the irradiated cells are incubated in distilled water or buffer for a number of hours prior to plating. This phenomenon is called liquid-holding recovery. We found that the liquid-holding procedure served not only to restore the viability of the irradiated cells, but also to alter their sensitivity to further doses of radiation. Specifically, the ultraviolet sensitivity of cells which had undergone liquid-holding recovery was markedly decreased, whereas their X-ray sensitivity appeared to be slightly increased. These sensitivity changes were qualitatively the same irrespective of whether the initial radiation exposure was to X rays or ultraviolet light. (In contrast, the radiation sensitivity of cells which had undergone maximal photoreactivation was essentially the same as that of untreated controls.) It is suggested that these changes in radiosensitivity are the result of structural alterations induced in the cell's deoxyribonucleic acid by the execution of at least the initial steps of a deoxyribonucleic acid repair process during the liquid-holding period.     

Protective Effect of Carvacrol on Oxidative Stress and Cellular DNA Damage Induced by UVB Irradiation in Human Peripheral Lymphocytes

Exposure to ultraviolet B (UVB; 280‐320 nm) radiation induces the formation of reactive oxygen species (ROS) in the biological system. In this study, we examined the protective effect of carvacrol on UVB‐induced lipid peroxidation and oxidative DNA damage with reference to alterations in cellular an‐tioxidant status in human lymphocytes. A series of in vitro assays (hydroxyl radical, superoxide, nitric oxide, DPPH (2,2‐Diphenyl‐1‐picryl hydrazyl), and ABTS (2,2‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid) radical scavenging assays) demonstrate antioxidant property of carvacrol in our study. UVB exposure significantly increased thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LHPs), % tail DNA and tail moment; decreased % cell viability and antioxidant status in UVB‐irradiated lymphocytes. Treatment with carvacrol 30 min prior to UVB‐exposure resulted in a significant decline of TBARS, LHP, % tail DNA, and tail moment and increased % cell viability as carvacrol concentration increased. UVB irradiated lymphocytes with carvacrol alone (at 10 μg/mL) gave no significant change in cell viability, TBARS, LHP, % tail DNA, and tail moment when compared with normal lymphocytes. On the basis of our results, we conclude that carvacrol, a dietary antioxidant, mediates its protective effect through modulation of UVB‐induced ROS.     

Karyometric observations of WISH cell cultures irradiated with 3 GHz microwaves.

WISH cell cultures 24 hours after passage were irradiated with 3 GHz microwaves (10 cm) at far field conditions in free space (anechoic chamber) for 30 minutes, at field power density 5 or 20 mW/cm2. Within 1,24 and 48 hours of the exposure to microwave fields the volumes of nuclei and nucleoli were measured with the use of a micrometer, and logvolumes and nucleo-nucleolar ratios were calculated. Under the applied irradiation conditions the culture medium temperature did not exceed 37 degrees C. In cultures irradiated at field power density 20 mW/cm2 increased number of cells with small nuclei and enlarged nucleoli was noted within 1 hour of the exposure. Within 24 and 48 hours after irradiation the nucleolar volume showed a slight decrease, whereas the nuclear volume increased. In cultures irradiated at field power density 5 mW/cm2 increased numbers of cells with enlarged nuclei and nucleoli were found. Analysis of the distribution curves of nuclear and nucleolar volumes suggests that non-thermal power densities of microwaves stimulate the metabolism of cell cultures. However, at higher power densities (20 mW/cm2) the stimulation phase is preceded by a period of reduced viability of cell cultures.     

Centrifugal separation of irradiated cultures of Escherichia coli cells into viable and nonviable populations.

Incubation of ultraviolet-irradiated Escherichia coli B/r cultures with 0.7% Triton X-100 resulted in a large decrease in turbidity. Under phase-contrast optics, most of the irradiated detergent-treated cells were smaller than normal and of low phase density; only a small percentage were normal or larger than normal and of normal phase density. Irradiated cells not treated with detergent showed fewer pronounced morphological changes. Irradiated cells treated with detergent lost large amounts of proteins and ribonucleic acid, but not of deoxyribonucleic acid. Such cultures could be separated by centrifugation into populations of (i) slowly sedimenting cells consisting of small, phase-light cells of low viability and (ii) large cells of normal phase density and high viability (100%). A similar separation was effected in gamma-irradiated cultures.     

A DNA excision repair system for Neisseria gonorrhoeae

Summary The removal of pyrimidine dimers from deoxyribonucleic acid of ultraviolet irradiated cultures of Neisseria gonorrhoeae can not be readily ascertained by using radioactively labeled thymidine percursors. However, by adapting the alkaline agarose gel technique of Achey et al. (Photochem Photobiol 29, 305–310, 1979), it was possible to demonstrate that this human pathogen does possess an active excision repair system that functions on pyrimidine dimers.This work was performed as partial fulfillment for a Doctoral Thesis by L.A. Campbell.     

紫外处理梨小食心虫卵对暗黑赤眼蜂寄生和羽化的影响

【目的】研究暗黑赤眼蜂Trichogramma pintoi Voegele对经紫外线照射处理的梨小食心虫 Grapholita molesta (Busck)卵的寄生效果, 确定处理寄主卵的最佳紫外强度和处理时间, 为利用小卵大量饲养赤眼蜂时寄主卵的处理和保存提供方法。【方法】初羽化12 h内的暗黑赤眼蜂分别寄生经不同强度紫外灯处理不同时间的梨小食心虫卵, 观察其寄生状况, 并统计寄生率和羽化率, 与对未经紫外处理的梨小食心虫卵的寄生率和羽化率作比较。【结果】暗黑赤眼蜂对经紫外照射的梨小食心虫卵的寄生率明显下降, 且随着紫外光强度的增强和紫外处理时间的延长, 影响强度增大。紫外处理梨小食心虫卵后, 暗黑赤眼蜂羽化率变化不大, 用15W紫外灯1-2 h或30W紫外灯照射1 h后, 暗黑赤眼蜂羽化率有所提高, 均在80%以上, 但在紫外照射3 h后, 羽化率明显下降。【结论】处理梨小食心虫卵时的紫外光强度及紫外处理时间对暗黑赤眼蜂寄生梨小食心虫卵的寄生效果均有一定的影响。实验室利用梨小食心虫卵大量繁殖暗黑赤眼蜂时, 宜采用15W 1 h紫外照射, 既能杀死寄主卵的胚胎, 又不会对暗黑赤眼蜂的寄生效果产生明显的不利影响。     

Bystander response in human lymphoblastoid TK6 cells

The mechanisms of the medium-mediated bystander response induced by γ-rays in non-irradiated TK6 cells were investigated. Cell cultures were irradiated and the culture medium discarded immediately after irradiation and replaced with a fresh one. In cells incubated with conditioned medium from irradiated cells (CM), a significant decrease in cell viability and cloning efficiency was observed, together with a significant increase in apoptosis, also in directly irradiated cells. To examine whether bystander apoptosis involved the extrinsic pathway, an inhibitor of caspase-8 was added to CM cultures, which significantly decreased apoptosis to control levels. The addition to CM of ROS scavengers, Cu–Zn superoxide dismutase and N-acetylcysteine did not affect the induction of apoptosis. To assess whether CM treatment activates a DNA damage response, also the formation of γ-H2AX foci, as markers of double-strand breaks and their colocalisation with 53-binding protein 1 (53BP1) and the protein mutated in the Nijmegen breakage syndrome 1 (NBS1) was analysed. In cultures treated for 2 h with CM, 9–11% of cells showed γ-H2AX foci, which partially or totally lacked colocalisation with 53BP1 and NBS1 foci. About 85% of irradiated cells were positive for γ-H2AX foci, which colocalised with 53BP1 and NBS1 proteins. At 24 h from irradiation, very few irradiated cells retained foci, fitting DNA repair kinetics. The number of foci-positive bystander cells also decreased to background values 24 h after CM incubation. Our results suggest that irradiated TK6 cells release into the medium some soluble factors, not ROS, which are responsible for the cytotoxic effects induced in bystander cells. In our experimental system, the role of ROS appeared to be of minor importance in inducing cell mortality, but probably critical in activating the DNA damage response in the responsive fraction of bystander cells.     

Evidence Relating Cessation of Respiration, Cell Envelope Changes, and Death in Ultraviolet-Irradiated Escherichia coli B/r Cells

Ionic and nonionic detergents have little effect on respiring bacteria, but in cultures poisoned with KCN rapid solubilization of the cell membrane, as indicated by turbidity losses, takes place. Ultraviolet radiations cause Escherichia coli cells grown in minimal medium with glycerol as a carbon source to cease respiring and growing about 1 h after irradiation. We tested the effect of the nonionic detergent Triton X-100 on growth and cell membrane dissolution (both measured by turbidity changes), respiration, and viability of unirradiated and irradiated E. coli B/r cells. When the detergent was added to cells immediately after irradiation, a decrease in turbidity occurred only when respiration was about to cease; when it was added after cessation of respiration, the turbidity loss was immediate. In both cases the turbidity loss was about 60%, and disintegration of the cell walls did not take place. 5-Fluorouracil (FU) and thermal (42 C) treatments cause respiration of irradiated cells to be maintained and also cause viability increases. Irradiated cells treated with FU and detergent show no turbidity loss just prior to the time respiration normally ceases, but a loss does occur in irradiated cells incubated with detergent at 42 C. We conclude that FU maintains respiration for all of the cells, but that thermal treatment maintains respiration for only part of the cells. In all cases the detergent had only a negligible effect on the respiration and viability of unirradiated and irradiated cells. We conclude that Triton X-100 causes solubilization of cell membranes of only nonrespiring cells that are not destined to survive.     

Effect of ursolic acid, a triterpenoid antioxidant, on ultraviolet-B radiation-induced cytotoxicity, lipid peroxidation and DNA damage in human lymphocytes

Exposure to ultraviolet B (UVB, 280-320) radiation induces the formation of reactive oxygen species (ROS) in the biological system. In this study, we examined the protective effect of ursolic acid on UVB-induced lipid peroxidation and oxidative DNA damage with reference to alterations in cellular antioxidant status in human lymphocytes. Series of in vitro tests (hydroxyl radical, superoxide, nitric oxide, DPPH and ABTS radical scavenging assays) demonstrates antioxidant property of ursolic acid in our study. Treatment of lymphocytes with ursolic acid alone (at 10 microg/mL) gave no significant change in cell viability, thiobarbituric acid reactive substances (TBARSs), lipid hydroperoxides (LHPs), % tail DNA and tail moment when compared with normal lymphocytes. UVB-exposure significantly increased TBARS, LHP and % tail DNA, tail moment; decreased % cell viability and antioxidant status in irradiated lymphocytes. Treatment with ursolic acid 30 min prior to UVB-exposure resulted in a significant decline of TBARS, LHP, % tail DNA and tail moment and increased % cell viability as ursolic acid concentration increased. Based on our results we conclude that ursolic acid, a dietary antioxidant, mediates its protective effect through modulation of UVB-induced reactive oxygen species.