Pyrimidine Dimers in the DNA of Paramecium aurelia

The production and fate of thymine-containing pyrimidine dimers in Paramecium aurelia DNA was investigated in three experimental series: production of dimers by UV irradiation, fate of dimers in the dark, and “loss of photoreactivability of dimers.” It is shown that cyclobutyl dimers are made by UV irradiation of Paramecium DNA in vivo, that because of cytoplasmic absorption the number of dimers made in DNA irradiated in vivo is much lower than in DNA irradiated in vitro, that dimers are lost from animals incubated in the dark after irradiation, and that all the dimers that remain in the animals can be destroyed by photoreactivating illumination. Since mutation induction is photoreactivable, these and previous photoreactivation data suggest that pyrimidine dimers are important in mutation induction in P. aurelia.     

Epifluorescent microscopic studies on the mechanism of preferential destruction of chloroplast nucleoids of male origin in young zygotes ofChlamydomonas reinhardtii

Summary The studies on the kinetics of nucleoid destruction reported here showed that destruction of chloroplast nucleoids (ct nucleoids) of male origin began to occur at about 30 minutes after mixing of male (mt^–) and female (mt⁺) gametes. The timing of initiation of the destruction differed among zygotes but usually occurred during 50–120 minutes after mixing. About 10 minutes was required for complete digestion of the ct nucleoids. UV irradiation on young zygotes or addition of an RNA-synthesis inhibitor, actinomycin D, to the incubation medium during the first 0–30 minutes after mixing almost completely inhibited the incorporation of³H uridine into the cell nuclei and the preferential destruction without inhibiting cell nuclear fusion. These results suggest that soon after mating,de novo RNA synthesis is concerned for the preferential destruction of ct nucleoids. To determine in which of the two cell nuclei in the zygotes the RNA is synthesized, each gamete (mt^–, mt⁺) was irradiated with UV and mated with unirradiated gametes of opposite mating type. This treatment of the male gametes had no effect on the incorporation of³H uridine into cell nuclei and the preferential destruction of ct nucleoids but UV irradiation of female gametes almost completely inhibited the incorporation of³H uridine into cell nuclei and the preferential destruction of ct nucleoids. Similar phenomena occurred in other crosses. The UV effect was photoreactivated in about 50% by white light, suggesting that the UV target is DNA. Thus, RNA synthesized in the cell nucleus of female origin soon after mating may be responsible for the preferential destruction of ct nucleoids of male origin     

The absence of Ser389 phosphorylation in p53 affects the basal gene expression level of many p53-dependent genes and alters the biphasic response to UV exposure in mouse embryonic fibroblasts

Phosphorylation is important in p53-mediated DNA damage responses. After UV irradiation, p53 is phosphorylated specifically at murine residue Ser389. Phosphorylation mutant p53.S389A cells and mice show reduced apoptosis and compromised tumor suppression after UV irradiation. We investigated the underlying cellular processes by time-series analysis of UV-induced gene expression responses in wild-type, p53.S389A, and p53^−/− mouse embryonic fibroblasts. The absence of p53.S389 phosphorylation already causes small endogenous gene expression changes for 2,253, mostly p53-dependent, genes. These genes showed basal gene expression levels intermediate to the wild type and p53^−/−, possibly to readjust the p53 network. Overall, the p53.S389A mutation lifts p53-dependent gene repression to a level similar to that of p53^−/− but has lesser effect on p53-dependently induced genes. In the wild type, the response of 6,058 genes to UV irradiation was strictly biphasic. The early stress response, from 0 to 3 h, results in the activation of processes to prevent the accumulation of DNA damage in cells, whereas the late response, from 12 to 24 h, relates more to reentering the cell cycle. Although the p53.S389A UV gene response was only subtly changed, many cellular processes were significantly affected. The early response was affected the most, and many cellular processes were phase-specifically lost, gained, or altered, e.g., induction of apoptosis, cell division, and DNA repair, respectively. Altogether, p53.S389 phosphorylation seems essential for many p53 target genes and p53-dependent processes.     

Effects of ultraviolet radiation on the membranes ofChara corallina

Summary The effects of 253.7 nm ultraviolet (UV) radiation on the membrane properties ofChara corallina have been studied. UV irradiation caused depolarization of the membrane potential (p.d.) and a decrease in membrane resistance. These effects were largely reversible with steady values being obtained within 40 minutes after the UV was turned off. The effects on ionic fluxes of Na⁺, K⁺ and Cl^– have also been studied using radioactive tracer techniques. The influxes were unchanged by irradiation. The chloride efflux was increased sevenfold during the irradiation period but recovered to the pre-irradiation value within 30 minutes after the irradiation period. The potassium efflux was also increased and reached a maximum 10 minutes after irradiation. The resting potential and the average depolarized p.d. reached during irradiation were in good agreement with those calculated from permeability coefficients indicated by the observed passive fluxes, using the Goldman equation for p.d. However, the plasmalemma resistance and its change due to irradiation did not match the values calculated from the same permeability coefficients used to estimate p.d. This disagreement, and an apparent imbalance in the charge transferred across the resting or irradiated plasmalemma, suggest the participation of another ion species as well as K⁺, Na⁺ and Cl^–.     

Comparison on the genotoxic effects of nuclear vs cytoplasmic irradiation from the alteration ofCD59 gene locus

Using microbeam to irradiate human-hamster hybrid A_L cells with defined number of α particles in a highly localized spatial region, this paper showed that cytoplasmic irradiation induced very little toxicity. For example, the cell killing by 4 α particle traversal through the cytoplasm was about 10%, and about 70% cells survived after their cytoplasm was irradiated with 32 a particles. In contrast, the survival fractions for nuclear irradiation at the same doses were 35% and less than 1% respectively. Mutation induction showed that while nuclear irradiation induced 3—4-fold more CD59^- mutants than cytoplasmic irradiation at equivalent particle traversal, at an equitoxic dose level of 90% survival, the latter exposure mode induced 3.3-fold more mutants than nuclear irradiation. Moreover, using multiplex PCR to analyze five marker genes on chromosome 11 (WT, CAT, PTH, APO-A1 and RAS), the results showed that the majority of mutants induced by cytoplasmic irradiation had retained all of the marker genes analyzed. By comparison, the proportion of mutants suffering loss of additional chromosomal markers increased with increasing number of particle traversal through nuclei.     

Postreplication Repair of Ultraviolet Damage to DNA, DNA-Chain Elongation, and Effects of Metabolic Inhibitors in Mouse L Cells

Alkaline sucrose sedimentation studies of DNA from mouse L cells have demonstrated the following effects of several inhibitors of nucleic acid and protein synthesis on postreplication repair of ultraviolet (UV) damage to their DNA. The DNA newly synthesized by a 2 h [³H]thymidine (dThd) label following 254 nm UV irradiation of 20 J/m² is made in smaller segments of the number average mol wt (Mn) of ~10 × 10⁶ than the control of ~40 × 10⁶. The presence of caffeine at a concentration of 2 mM during the labeling of the irradiated cells reduces the Mn value to 5.8 × 10⁶, which is nearly comparable to, but somewhat larger than the expected distance between dimers in parental DNA. Afterwards, such an interrupted DNA made in the irradiated cells is completely repaired to the present maximum Mn value of 40 × 10⁶ in the consecutive 4 h chase in unlabeled dThd. The presence of the nucleic acid inhibitor, either 2 mM hydroxyurea, 50 μM arabinofuranosyl cytosine, 2 mM excess dThd or 5 μg/ml of actinomycin D (AMD) during 2- to 24-h chase periods after a 2 h postirradiation label prevents the repair to various extents, while 2 mM caffeine completely inhibits it. In the unirradiated cells, these agents except excess dThd and caffeine also interfere severely with normal elongation of nascent DNA made by a 3 min pulse label, but do not appreciably induce single chain breaks of either newly synthesized or parental DNA. The inhibition of the repair by AMD suggests that de novo elongation of DNA to close the gaps in new DNA made in the irradiated cells requires at least a template-dependent DNA polymerase. In contrast, 100 μg/ml of cycloheximide allows to complete the gap-filling repair, while it simply reduces the rates of chain growth for the repair and normal replication. Therefore, the similar sensitivity of gap-filling repair and normal replication towards the above inhibitors indicates that a preexisting DNA polymerizing system appears to be responsible and to play a common role without new protein synthesis, as far as the repair at early time after UV is concerned.     

Reactivation of Herpes Simplex Virus Type 1 in the Mouse Trigeminal Ganglion: an In Vivo Study of Virus Antigen and Cytokines

Reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG) was induced by UV irradiation of the corneas of latently infected mice. Immunocytochemistry was used to monitor the dynamics of cytokine (interleukin-2 [IL-2], IL-4, IL-6, IL-10, gamma interferon [IFN-γ], and tumor necrosis factor alpha [TNF-α]) and viral antigen production in the TG and the adjacent central nervous system on days 1 to 4, 6, 7, and 10 after irradiation. UV irradiation induced increased expression of IL-6 and TNF-α from satellite cells in uninfected TG. In latently infected TG, prior to reactivation, all satellite cells were TNF-α⁺ and most were also IL-6⁺. Reactivation, evidenced by HSV-1 antigens and/or infiltrating immune cells, occurred in 28 of 45 (62%) TG samples. Viral antigens were present in the TG in neurons, often disintegrating on days 2 to 6 after irradiation. Infected neurons were usually surrounded by satellite cells and the foci of immune cells producing TNF-α and/or IL-6. IL-4⁺ cells were detected as early as day 3 and were more numerous by day 10 (a very few IL-2⁺ and/or IFN-γ⁺ cells were seen at this time). No IL-10 was detected at any time. Our observations indicate that UV irradiation of the cornea may modulate cytokine production by satellite cells. We confirm that neurons are the site of reactivation and that they probably do not survive this event. The predominance of TNF-α and IL-6 following reactivation parallels primary infection in the TG and suggests a role in viral clearance. The presence of Th2-type cytokines (IL-4 and IL-6) indicates a role for antibody. Thus, several clearance mechanisms may be at work.     

Effect of ultraviolet radiation on cell division and microtubule organization inPetunia hybrida protoplasts

Summary Mesophyll protoplasts isolated fromPetunia hybrida were subjected to UV radiation (280–360 nm) in an attempt to assess whether (a) UV radiation has an effect on cortical microtubule organization, (b) UV radiation affects the progression of protoplasts through the cell cycle, and (c) there is a connection between the effect of UV radiation on cell division and the polymerization state of the microtubules. The proto plasts were irradiated with the following UV doses: 4, 8, 12, and 24mmol photons/m², 30 min after isolation. Cell cycle analysis and immuno-localization of microtubules were carried out 0, 24, 48, and 72 h after irradiation. The length of cortical microtubules was determined after irradiation and in corresponding controls. We found that UV radiation induced breaks in cortical microtubules resulting in shorter fragments with increasing dose. Also, the protoplasts were delayed in their progression through the cell cycle, with G1 and G2 phases being affected as well as the S phase. The commencement of DNA synthesis in the irradiated protoplasts followed the re-establishment of a microtubule network. At 48 h after irradiation the protoplasts in all treatments, except for the 24 mmol/m², had cortical microtubules of similar length, and at 72 h after irradiation only the protoplasts that had received 24 mmol photons/m² had not started dividing.Abbreviations BSA bovine serum albumin - DMSO dimethyl sulfoxide - FDA fluorescein diacetate - MT microtubules - MTSB microtubule stabilizing buffer - PAR photosynthetically active radiation (400–700 nm) - PBS phosphate buffered saline - UV ultraviolet     

Genomic landscape of single-stranded DNA gapped intermediates in Escherichia coli

Single-stranded (ss) gapped regions in bacterial genomes (gDNA) are formed on W- and C-strands during replication, repair, and recombination. Using non-denaturing bisulfite treatment to convert C to U on ssDNA, combined with deep sequencing, we have mapped gDNA gap locations, sizes, and distributions in Escherichia coli for cells grown in mid-log phase in the presence and absence of UV irradiation, and in stationary phase cells. The fraction of ssDNA on gDNA is similar for W- and C-strands, ∼1.3% for log phase cells, ∼4.8% for irradiated log phase cells, and ∼8.5% for stationary phase cells. After UV irradiation, gaps increased in numbers and average lengths. A monotonic reduction in ssDNA occurred symmetrically between the DNA replication origin of (OriC) and terminus (Ter) for log phase cells with and without UV, a hallmark feature of DNA replication. Stationary phase cells showed no OriC → Ter ssDNA gradient. We have identified a spatially diverse gapped DNA landscape containing thousands of highly enriched ‘hot’ ssDNA regions along with smaller numbers of ‘cold’ regions. This analysis can be used for a wide variety of conditions to map ssDNA gaps generated when DNA metabolic pathways have been altered, and to identify proteins bound in the gaps.     

Dependence of the U.V. Survival of transforming DNA on the amount of DNA uptake per cell

Summary UV irradiation of transforming DNA from Haemophilus influenzae, carrying a streptomycin resistance marker (Sr), results in decreased transforming activity. At high DNA concentration the marker survival is lower than it is at low concentration. The transition from high to low survival occurs at concentrations ranging from 2.5×10^-3 to 2.5×10^-2 g/ml; in this range the probability that transformed cells take up DNA fragments in addition to the marked one increases rapidly. A similar effect of DNA concentration on the percentage of transformants is observed for a mixture of unirradiated and irradiated DNA, where virtually all of the transformants originate from the unirradiated component. This eliminates the possible explanation that the concentration dependence of UV survival of a marker reflects increasing competition for a cellular repair system.It is concluded that the lower marker survival obtained at high DNA concentration involves lethality due to UV lesions present in the additional irradiated DNA taken up by the cell. Thus the steeper marker survival curve is due to the increasing UV dose which the additional DNa necessarily receives when a marker survival curve is being established. Intergration of UV lesions rendering a chromosomal DNA strand inviable is suggested by a slight delay in cell multiplication after uptake of irradiated and — to a lesser extent — unirradiated DNA. Acriflavine at a concentration of 0.5g/ml enhances the effect of DNA concentration on marker survival. Similarly the number of transformants obtained with unirradiated DNA in the presence of acriflavine is more strongly decreased at high than at low DNA concentration. It is suggested that each event of DNA integration involves a small change for lethality, which is enhanced if the DNA carries UV lesions or if acriflavine is present.Dedicated to Professor H. Nachtscheim on the occasion of his 80th birthday.     

Analysis of Minimal Functions of Simian Virus 40 II. Enhancement of Oncogenic Transformation in Vitro by UV Irradiation

After light UV irradiation (5,000 to 10,000 ergs/mm²) “complete” and “defective” simian virus 40 (SV40) showed an enhancement of oncogenic transformation capacity in Syrian hamster kidney cells in vitro up to 180 and 270% of the controls, respectively. Simultaneously with the enhancement of transformation, an increase in T-antigen induction was observed in CV-1 cells infected with light UV-irradiated SV40; infectivity, however, was correspondingly reduced by 1 log₁₀. After strong UV irradiation (10,000 to 80,000 ergs/mm²) of “complete” and “defective” SV40, transformation capacity in vitro proved to be the most resistant viral function. It was only slightly reduced in comparison with a 4 to 5 log₁₀ reduction of infectivity. T-antigen induction of SV40 was also equally resistant to strong UV irradiation. We found no evidence of “multiplicity reactivation” involved in the high resistance of transformation capacity of SV40 after UV irradiation. Syrian hamster kidney cells transformed in vitro by UV-irradiated SV40 contained the SV40-specific T-antigen and showed the same morphology and growth characteristics as cells transformed by non-irradiated “complete” or “defective” SV40. They induced malignant tumors after subcutaneous inoculation into Syrian hamsters.     

UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2±0.2 × 10^–10 cm²/s, and 1.8±0.2 × 10^–10 cm²/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3±0.3 × 10^–10 cm²/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1±0.8 × 10^–10 cm²/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.Abbreviations CAT Catalase - D Lateral diffusion coefficient - EDTA Ethylenediaminetetraacetic acid - EGF Epidermal growth factor - E-MEM Eagle's minimum essential medium - FCS Fetal calf serum - FRAP Fluorescence recovery after photobleaching - KRG Krebs-Ringer phosphate buffer - PBS Phosphate-buffered saline - R Mobile fraction - ROS Reactive oxygen species - SEM Standard error of the mean - SOD Superoxide dismutase - UVA Ultraviolet light-A (315-400 nm) - UVB Ultraviolet light-B (280-315 nm)     

Inducible reactivation of UV-irradiated cholera phage e5 in Vibrio cholerae MAK757

Summary The survival of UV-irradiated cholera phage e5 was found to increase when the host cells, Vibrio cholerae MAK757, were exposed to a low dose of UV irradiation before phage infection (Weigle reactivation), indicating the existence of a UV-inducible DNA repair pathway (SOS repair) in V. cholerae MAK757. The induction signal generated by UV irradiation was transient in nature and lasted about 20–30 min at 37°C. Maximal weigle reactivation of the phage was obtained when the host cells were irradiated with a UV dose of 16 J/m². V. cholerae MAK757 was also found to possess efficient photoreactivation and host cell reactivation of UV-damaged DNA in phage e5.     

Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation

Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G₀ state. Autoradiography studies of cells released from G₀ after 72 h and replated at lower densities (3?9 × 10³ cells/cm²) in fresh medium containing 15% fetal bovine serum showed that semiconservative DNA synthesis (S phase) began ～24 h after the replating. To determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts, we released cultures of NF or XP12BE cells from G₀, allowed them to reattach at lower densities, irradiated them in early G₁ (～18 h prior to the onset of S) or just prior to S phase, and assayed the frequency of mutations to 6-thioguanine resistance and the survival of colony-forming ability. The XP12BE cells, which are virtually incapable of excising UV-induced DNA lesions, showed approximately the same frequency of mutations and survival regardless of the time of UV irradiation. In NF cells, the slope of the dose response for mutations induced in cells irradiated just prior to S was about 7-fold steeper than that of cells irradiated 18 h earlier. However, the two sets of NF cells showed no significant difference in survival. Neither were there significant differences in the survival of NF cells released from G₀, plated at cloning densities and irradiated as soon as they had attached and flattened out (～20 h prior to S) or 4, 8, 12, 16, 20 or 24 h later. We conclude that the frequency of mutations induced by UV is dependent upon the number of unexcised lesions remaining at the time of semi-conservative DNA replication. However, the amount of time available for excision of potentially cytotoxic lesions is not determined primarily by the period between irradiation and the onset of S phase.     

Dependence of DNA dark repair on protein synthesis in Escherichia coli.

Summary We investigated the influence of aminoacidless treatments applied prior and after UV irradiation on survival, dimer excision, postirradiation DNA degradation, DNA synthesis and sedimentation profiles of parental DNA ofE. coli B/r Hcr⁺ cells. In dependence on the treatment applied, the fluence 50 J/m² yielded distinctly different fractions of survivors within 0,03–85%. In all cases dimers were completely excised. The rate of DNA degradation was similar during a 30–40 min period after UV during which the bulk of dimers was excised. Degradation ceased, however, earlier in the prestarved cells than in exponentially growing ones; it was prolonged by aminoacidless postincubation. Sedimentation profiles of parental DNA did not differ during the whole period of dimer excision. In cells DNA synthesis was not restored for several hours after addition of amino acids. In cells addition of amino acids resulted in a fast resumption of DNA synthesis. We conclude that removal of dimers and repair of gaps were similar in all cases. We believe that aminoacidless treatments influence production and repair of damage to the sites of DNA replication. The treatment appears to prevent this damage when applied before UV irradiation, but interferes with its restoration when applied after UV irradiation. Consequently, the former treatment increases survival of cells while the latter produces an opposite effects.     

Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells

Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×10⁶ cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF₅₀ and DMF₃₇) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective “off the shelf” therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia.     

Inhibition of mitochondria-dependent apoptosis by 635-nm irradiation in sodium nitroprusside-treated SH-SY5Y cells

Nitric oxide (NO) is a major factor contributing to the loss of neurons in ischemic stroke, demyelinating diseases, and other neurodegenerative disorders. NO not only functions as a direct neurotoxin, but also combines with superoxide (O₂⁻) by a diffusion-controlled reaction to form peroxynitrite (ONOO⁻), a species that contributes to oxidative signaling and cellular apoptosis. However, the mechanism by which ONOO⁻ induces apoptosis remains unclear, although subsequent formation of reactive oxygen species (ROS) has been suggested. The aim of this study was to further investigate the triggers of the apoptotic pathway using O₂⁻ scavenging with light irradiation to block ONOO⁻ production. Antiapoptotic effects of light irradiation in sodium nitroprusside (SNP)-treated SH-SY5Y cells were assayed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DNA fragmentation, flow cytometry, Western blot, and caspase activity assays. In addition, NO, total ROS, O₂⁻, and ONOO⁻ levels were measured to observe changes in NO and its possible involvement in radical induction. Cell survival was reduced to approximately 40% of control levels by SNP treatment, and this reduction was increased to 60% by low-level light irradiation. Apoptotic cells were observed in the SNP-treated group, but the frequency of these was reduced in the irradiation group. NO, O₂⁻, total ROS, and ONOO⁻ levels were increased after SNP treatment, but O₂⁻, total ROS, and ONOO⁻ levels were decreased after irradiation, despite the high NO concentration induced by SNP treatment. Cytochrome c was released from mitochondria of SNP-treated SH-SY5Y cells, but not of irradiated cells, resulting in a decrease in caspase-3 and -9 activity in SNP-treated cells. Finally, these results show that 635-nm irradiation, by promoting the scavenging of O₂⁻, protected against neuronal death through blocking the mitochondrial apoptotic pathway induced by ONOO⁻ synthesis.     

Cellular kinetics of the peripheral nerve and striated muscle after a single dose of X-rays

Summary Thirtysix mice were irradiated with a single dose of X-rays (2760 rad) on one leg, while the rest of the body was shielded by lead. The animals were killed 1–270 days after the irradiation and ³H-thymidine was injected 2 hours before death. Sections from the sciatic nerve and its innervated muscles were examined by autoradiography and by special methods for the mast cells. No changes in the endoneurial mast-cell component could be disclosed after irradiation. At all times after the irradiation, the numbers of labelled Schwanncell nuclei and the sarcolemna-cell nuclei were moderately decreased on the irradiated side, when compared with the control side.Supported by a grant from the Swedish Medical Research Council.     

The involvement of nuclear nucleases in rat thymocyte DNA degradation after γ-irradiation

Possible mechanisms of internucleosomal DNA fragmentation in thymocytes of irradiated rats were studied. It was shown that thymocyte nuclei contain at least two nucleases that cleave DNA between nucleosomes — a Ca²⁺/Mg²⁺-dependent nuclease and an acidic one which does not depend on bivalent ions. 2 and 3 h after irradiation at a dose of 10 Gy the initial rate of DNA cleavage by Ca²⁺/Mg²⁺-dependent nuclease in isolated nuclei increased three and seven times, respectively, but the kinetics of DNA digestion by acidic nuclease did not change. The experiments with cycloheximide indicated that Ca²⁺/Mg²⁺-dependent endonuclease turns over at a high rate. The activity of the cytoplasmic acidic and Mg²⁺-dependent nucleases was shown to increase (by 40 and 50%, respectively) 3 h after irradiation. The effect is caused by the de novo synthesis of the nucleases. At the same time the activity of nuclear nucleases did not essentially change. The chromatin isolated from rat thymocytes 3 h after irradiation did not differ in its sensitivity to some exogenic nucleases (DNAase I, micrococcal nuclease and nuclease from Serratia marcescens) from the control. Thus, Ca²⁺/Mg²⁺-dependent endonuclease seems to be responsible for the postirradiation internucleosomal DNA fragmentation in dying thymocytes.     

Experimental set up for the irradiation of biological samples and nuclear track detectors with UV C

Aim

In this work we present a methodology to produce an “imprint” of cells cultivated on a polycarbonate detector by exposure of the detector to UV C radiation.

Background

The distribution and concentration of ¹⁰B atoms in tissue samples coming from BNCT (Boron Neutron Capture Therapy) protocols can be determined through the quantification and analysis of the tracks forming its autoradiography image on a nuclear track detector. The location of boron atoms in the cell structure could be known more accurately by the simultaneous observation of the nuclear tracks and the sample image on the detector.

Materials and Methods

A UV C irradiator was constructed. The irradiance was measured along the lamp direction and at different distances. Melanoma cells were cultured on polycarbonate foils, incubated with borophenylalanine, irradiated with thermal neutrons and exposed to UV C radiation. The samples were chemically attacked with a KOH solution.

Results

A uniform irradiation field was established to expose the detector foils to UV C light. Cells could be seeded on the polycarbonate surface. Both imprints from cells and nuclear tracks were obtained after chemical etching.

Conclusions

It is possible to yield cellular imprints in polycarbonate. The nuclear tracks were mostly present inside the cells, indicating a preferential boron uptake.