FORMATION OF NON-VIABLE SPORES OF DICTYOSTELIUM DISCOIDEUM BY UV-IRRADIATION AND CAFFEINE

Abstract We have characterized the spores formed from amoeboid cells of the wild type strain of Dictyostelium discoideum after UV-irradiation. Cell differentiation in the presence of caffeine after a fluence of 300 J/m² resulted in a population of spores which was 98% non-viable. The UV-irradiation did not affect the conversion of the spores to swollen spores but did affect the conversion of swollen spores to amoeboid cells. When the germination of the spores was done without caffeine, we detected only a small effect on conversion of swollen spores to amoeboid cells and on the beginning of growth. On the other hand, in the presence of caffeine, the spores had a remarkable delay in both. It was also shown that few, if any, pyrimidine dimers exist in the DNA of the non-viable spores. Possible mechanisms of formation of non-viable spores are discussed.     

ULTRAVIOLET EFFECTS ON KILLING, FRUITING BODY FORMATION AND THE SPORES OF DICTYOSTELIUM DISCOIDEUM

Abstract— -Ultraviolet effects on amoeboid cells of three strains of Dictyosrrlium discoidruin , NC-4. γs-13 and γs-18. for killing. fruiting body formation, spore formation and viability of the spores were studied.
The strain of γs-13 was more sensitive to UV light for killing than NC-4 at 10% survival. In addition. γs-13 was the most sensitive strain among the three for fruiting body formation and spore formation. The developmental process of this organism, however, showed a large resistance to UV light when compared with the killing. The spores of γs-13 formed after UV irradiation were mostly non-viable, though those of γs-18 and NC-4 were fully viable     

PHOTOTAXIS IN DICTYOSTELIUM DISCOIDEUM AMOEBAE

Abstract— An apparatus has been developed to measure phototactic movement in a population of amoebae of Dictyostelium discoideum. Fluence–response curves in white light show a positive phototaxis to light below 100mW/m². Higher intensities cause a negative phototaxis. An action spectrum, based on the zero-crossing points in fluence–response curves for monochromatic light, shows a major peak at about 405nm and secondary maxima at about 450, 520, 580 and 640nm. This action spectrum resembles the action spectra for accumulations of amoebae in and dispersal from light traps and that of inhibition of aggregation by light, but is distinctly different from the action spectrum for phototaxis by D. discoideum pseudoplasmodia.     

LIGHT-INDUCED ACCUMULATIONS OF DICTYOSTELIUM DISCOIDEUM AMOEBAE

Abstract— Light-induced accumulations of amoebae of Dictyostelium discoideum in 'light traps' have been observed. The greatest accumulations are obtained with cell densities of about 8 × 10⁴ cells/mm². Accumulations are observed at incident fluence rates over about one decade both for white and for monochromatic light; higher fluence rates cause dispersal from the 'light trap'. An action spectrum for the photoaccumulation, calculated from fluence-response curves using the zero thresholds, shows a major peak between 405 and 410 nm and extends through most of the visible spectrum. This action spectrum does not coincide with any of the pigments known to be present in D. discoideum . The cellular basis for the photoaccumulation has been studied. No light effects on cell divisions or cell aggregation are observed during the 2 h duration of an accumulation experiment. Microvideographic analysis of single amoebae is consistent with the hypothesis that the amoebae are positively phototactic and move toward the light scattered from cells in the 'light trap', thus accumulating in the trap.     

EFFECTS OF CAFFEINE ON DNA REPAIR OF UV-IRRADIATED DICTYOSTELIUM DISCOIDEUM

Abstract— Caffeine enhances the UV-killing of amoeboid cells of NC-4, but UV-irradiated γs-13 is insensitive to caffeine. UV-irradiated NC-4 becomes insensitive to the effect of caffeine during a postirradiation incubation in buffer for about 90 min, but γs-13 remains unchanged in the sensitivity to caffeine throughout the incubation for 180 min. Amoeboid cells of γs-13 can remove pyrimidine dimers as well as NC-4 even in the presence of caffeine. Caffeine inhibits rejoining of strand-breaks of DNA in UV-irradiated NC-4, but the rejoining in γs-13 is insensitive to caffeine.     

PHYSIOLOGICAL EFFECTS OF ULTRAVIOLET LIGHTON DICTYOSTELIUM DISCOIDEUM SPORE GERMINATION

Abstract— The spore germination in Dictyostelium discoideum consists of four stages: activation, postactivation lag, swelling and emergence. Ultraviolet irradiation (total fluence of 250 J/m²) of spores at any time prior to late spore swelling allows full swelling, but inhibits the emergence of myxamoebae. In the case of freshly activated spores, a UV exposure time of 30 s (total fluence of 50 J/m²) is sufficient to reduce emergence to about 6% when measured after 24 h of incubation. This same fluence results in about 10% viability as measured by plaque forming ability. Experiments utilizing 'fractionated exposures' result in the same percentage inhibition of emergence as that found for 'single exposures' provided the total fluence is equivalent. The higher fluences (250 J/m²) which completely prevent emergence, do not affect the endogenous oxygen uptake of spores during swelling. Ultraviolet light irradiated spores respond to the same activation and deactivation treatments as control unirradiated spores. Ultraviolet irradiation after late spore swelling allows emergence to occur in only a small fraction of the population. This fraction of cells which can emerge after UV treatment is said to have passed a 'competence point', which is believed to be the time when all the events necessary for emergence have been completed. Though the sites of UV inactivation in spores can only be postulated at present, it is apparent that the initial stages of germination (activation, postactivation lag and spore swelling) occur independently of the UV sensitive sites. The final stage of germination (emergence), however, is dependent on UV sensitive functions.     

INHIBITION OF UV-INDUCED DNA STRAND BREAKAGE IN DICTYOSTELIUM DISCOIDEUM BY 2,4–DINITROPHENOL

Abstract— In UV-irradiated vegetative cultures of the cellular slime mold Dictyostelium discoideum NC-4, single strand breaks appeared in the DNA very rapidly and at low temperatures (0–4°C). However, when these cells were incubated, prior to UV irradiation, in the presence of 2 m M 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, breaks did not appear in the DNA. Extracts prepared from cells that had been incubated either in the presence or absence of DNP were tested for endonucleolytic activities on a UV-irradiated exogenous DNA template (φX-174 RF I). Results suggested that DNP might mediate its effect by interfering with the action of a UV-specific endonuclease.     

EVENTS IN THE PHYTOCHROME MOLECULE AFTER IRRADIATION

The photoconversion of Pr to Pfr has been investigated by a large number of investigators. We have previously demonstrated that Z, E isomerization of the tetrapyrrole chromophore is involved in the photoconversion. It is the best candidate for the primary photoreaction. Conformation and configuration of the Pr chromophore will be compared with that of chromophores in phycocyanin. The crystal structure of phycocyanin had been elucidated by x-ray analysis. Proton transfer and/or Z, E isomerization of the tetrapyrrole are probably involved in different steps of the photoconversion in phytochrome and in photoreversible phycobiliproteins. Fluorescence decay kinetics of irradiated Pr and intermediate formation show heterogeneity. Possible reasons for this heterogeneity will be discussed.     

ULTRAVIOLET LIGHT-INDUCED INHIBITION OF CELL DIVISION AND DNA SYNTHESIS IN AXENICALLY GROWN REPAIR MUTANTS OF DICTYOSTELIUM DISCOIDEUM

Cell division and DNA synthesis were studied during axenic growth following 254 nm ultraviolet light (UV) irradiation of a repair-proficient parental strain ( rad⁺ , D₁₀ colony formation = 195 J/m²) and two repair mutants ( rad C. D₁₀= 50 J/m²; rad B. D₁₀= 5 J/m²) of Dictyostelium discoideum. Isopycnic CsCI gradients were used to distinguish uptake of labeled precursors into nuclear (n) and mitochondrial (m) DNA, using Netropsin to enhance the density resolution. In all strains, m-DNA synthesis was inhibited to a lesser extent than was n-DNA synthesis. For rad C, which has been shown in other experiments to be slow in incision and dimer removal, the UV-induced lags in division and n-DNA synthesis were longer than for rad⁺. However, rad B showed a more complex response. Although brief division lags were observed for < 10 J/m², little immediate division lag was detected at greater fluences. Instead, a brief period of cell multiplication of up to but not exceeding two-fold occurred, followed by a cessation of division, and then by lysis. Fluences that yielded extensive lags in n-DNA synthesis in rad^- and rad C resulted in little detectable immediate postirradiation lag in n-DNA synthesis in rad B. However, later in the postirradiation period, when DNA synthesis had resumed in rad⁺ and rad C. it gradually declined to near zero in rad B. We conclude: (1) that the more extended lag in division and n-DNA synthesis in rad C is consistent with its slower rate of excision repair, and (2) that rad B contains a defect resulting in less initial blockage of DNA replication by UV lesions.     

ENHANCEMENT OF ULTRAVIOLET OR N-METHYL-N'- NITRO-N-NITROSOGUANIDINE SENSITIVITY OF DICTYOSTELIUM DISCOIDEUM BY 3-AMINOBENZAMIDE

Abstract— The amoeboid cells of Dktyostelium discoideum NC–4 possess a 3-aminobenzamide(3-ABA)-sensitive repair mechanism for DNA damages induced by UV-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treatment. We have studied the effect of 3-ABA on each step of excision repair in the UV- irradiated cells. Although the nicking of DNA-strand and the excision of pyrimidine dimcrs are insensitive to 3-ABA, the rejoining of DNA strand-breaks is sensitive. The frequency of mutation induced by UV-irradiation or MNNG-treatment is depressed by 3-ABA. The mechanisms of repair inhibition by 3-ABA are discussed.     

FAR UV IRRADIATION OF DNA IN THE PRESENCE OF PROTEINS, AMINO ACIDS OR PEPTIDES

Abstract— The DNA of bacteriophage SP02c12 was subjected to 254 nm irradiation in solutions containing lysozyme or histone. In these solutions, the protein-DNA mass ratios and the ionic strengths of the solvents were varied to change the amount of protein associated with the DNA. Lysozyme-DNA binding constants were measured under the same conditions. The sensitivity of phage DNA to biological inactivation by UV increased as the amount of lysozyme bound per DNA strand increased. Although binding constants could not be measured for the DNA-histone interaction, this protein had a protective effect which was greater under conditions which cause enhanced binding. No crosslinking of either protein could be detected even at doses ten-fold greater than those giving a surviving fraction of 0.01.
Irradiation was also performed in the presence of various amino acids and short peptides. These were chosen to include amino acids which: (1) are positively charged, (2) absorb UV of this wavelength or (3) form UV-induced crosslinks to DNA. None of the amino acids tested affected sensitivity of the DNA to biological inactivation. Peptides containing a UV-absorbing amino acid and a positively charged amino acid enhanced sensitivity. For each of these peptides, a mixture of the constituent amino acids had the same effect as the peptide itself. Under the conditions used, no evidence for formation of DNA-amino acid crosslinks was found. The results indicate that proteins and peptides can sensitize DNA to UV inactivation by mechanisms other than covalent crosslink formation. Such mechanisms could include energy or electron transfer or alterations in the conformation of the DNA.     

EFFECTS OF RHODAMINE 123 IN THE DARK AND AFTER IRRADIATION ON MITOCHONDRIAL ENERGY METABOLISM

Isolated rat liver mitochondria have been used to study the mechanism of toxicity of Rhodamine 123 (Rho 123) in the dark and after irradiation with visible light. We report an inhibition of adenosine 5'-diphosphate phosphorylation which is increased after illumination. In the dark, the first steps of the phosphorylation process (i.e. the entry of substrates into the matrix, the electron transport to oxygen and the creation of the proton gradient) as well as ATPase activity are not significantly perturbed at Rho 123 concentration below 10 micrograms/mL. In contrast, the movements of the phosphate compounds are drastically impaired. Irradiation strengthens the detrimental effects in an oxygen dependent process. The nature of the noxious transient species is not clearly established, but it is suggested that singlet oxygen could be responsible for the observed damage.     

SURVIVAL, SPORE FORMATION AND EXCISION REPAIR OF UV-IRRADIATED DEVELOPING CELLS OF DICTYOSTELIUM DISCOIDEUM NC-4

Abstract— The synchronously developing aggregates of the cellular slime mold, Dictyostelium discoideum NC-4, were disaggregated into individual cells and irradiated with 254 nm UV light at preaggregation (0h), late interphase (6h), late aggregation (12 h), and preculmination (18 h). When assayed for replica-tive ability (colony formation), the developing cells at 0, 6, 12, and 18h showed the same sensitivity as vegetative cells; the 10% survival dose (D₁₀) was 160 J/m². The spores were more sensitive, with D₁₀ of 70 J/m². Excision repair of the nuclear DNA of the developing cells was studied by alkaline sucrose gradients. UV-induced single-strand breakage and rejoining of the DNA occurred to the same final extent in the cells from the 0, 6, 12 and 18 h stages of development, but a longer time was required for the completion of rejoining at the later stages (for example, at 54 J/m², 6.6 h for preculmination cells, 3.3 h for preaggregation cells). When the cells irradiated at various stages were required to redevelop, as measured by the relative numbers of spores produced, their sensitivity for completing this development increased the later the stage from which they were taken. The D₁₀s for spore production were 200, 130, 100 and 70 J/m² for cells at the 0, 6, 12 and 18 h stages, respectively. The fractional viability among the spores that appeared after this treatment was the same independent of the stage at which the cells were irradiated; the D₁₀ for this viability was 160 J/m², the same as if the cells had been plated immediately with no intervening developmental sequence. We conclude that DNA excision repair as related to replicative ability is retained at all stages of development; however, development seems independent of replicative ability and depends upon DNA and/or non-DNA damage in a more complex way.     

THE CHEMICAL TRANSFORMATION OF THE CATECHOLAMINES INDUCED BY UV IRRADIATION*

Abstract— The conversion of catecholamines exposed to ultraviolet irradiation has been investigated spectrophotometricaly. The UV activation of the molecule is a direct effect and the activation is not dependent upon the presence of metals. The transformation, which includes mdolization and polymerization requkes the presence of oxygen. The rate of the oxidative transformation is of the same order of magnitude in 100 per cent oxygen as in air, provided a metal chelating agent is present.
At neutral pH photoactivated adrenaline is oxidized to adrenochrome and further polymerized to melanin. At alkaline pH U.V. irradiation results in the formation of conversion products of adrenaline, different from those observed at neutral pH, and the melanin production is low.
When noradrenaline is subjected to U.V. irradiation at neutral pH the processes of oxidation and polymerization takes place at a much slower rate than that observed for adrenaline. The radiation effect on noradrenaline at alkaline pH is characterized by increased melanin formation.
Radicals derived from U.V. irradiation of H₂O₂ convert the catecholamines to different products, and no melanin formation can be recorded.
The sequence of reactions leading from the activated catecholamines to indolic structures, and the participation of the indolic structures in melanogenesis from the catecholamines is discussed.     

THE REGENERATION OF VISUAL PIGMENTS AND THE CHANGE OF ROD HYPERSENSITIVITY AFTER IRRADIATION BY BLEACHING LIGHT IN FROG RETINA

Abstract— The regeneration processes of visual pigments and the dark adaptation processes of rod photoreceptor after irradiation by bleaching light were studied by spectrophotometric, electroretinographic(ERG) methods and the measurement of early receptor potentials (ERPs) in bullfrog retina. After irradiation by bleaching light, rhodopsin in the isolated retina regenerated to an extent depending on the wavelength and intensity of the bleaching light as well as pH. Intense blue light and a weak alkaline environment (pH 7.5–9.5) favoured the regeneration. The regeneration of pigment in the green rods could not be detected in these experiments on the isolated retina. The regeneration of cone pigment was studied by measuring ERPs from both isolated retinas and retinas with pigment epithelium-choroid complex separated from scleras, which are called PEC-retinas. In the PEC-retinas, cone pigment regenerated more rapidly and with better efficiency than in the isolated retinas.
Rod photoreceptors desensitized permanently by bleaching light did not demonstrate hypersensitivity at 0.1 m M [Ca²⁺]_out, which induced hypersensitivity in non-desensitized photoreceptor, but showed the hypersensitivity when the [Ca²⁺]_out, was lowered further by the addition of EGTA.     

SINGLE-STRAND BREAKS IN THE DNA OF THE uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 AFTER ULTRAVIOLET IRRADIATION

Abstract— DNA single-strand breaks were produced in uvrA and uvrB strains of E. coli K-12 after UV (254 nm) irradiation. These breaks appear to be produced both directly by photochemical events, and by a temperature-dependent process. Cyclobutane-type pyrimidine dimers are probably not the photoproducts that lead to the temperature-dependent breaks, since photoreactivation had no detectable effect on the final yield of breaks. The DNA strand breaks appear to be repairable by a process that requires DNA polymerase I and polynucleotide ligase, but not the recA, recB, recF, lexA 101 or uvrD gene products. We hypothesize that these temperature-dependent breaks occur either as a result of breakdown of a thermolabile photoproduct, or as the initial endonucleolytic event of a uvrA , uvrB -independent excision repair process that acts on a UV photoproduct other than the cyclobutane-type pyrimidine dimer.     

PYRIMIDINE DIMER FORMATION AND GERMINATION OF UV-IRRADIATED SPORES OF DICTYOSTELIUM DISCOIDEUMNC–4 ANDys–13

Abstract— Survival, UV-photoproducts and germination of UV-irradiated spores of Dictyostelium discoi-deum were studied on two strains,NC–4 andys–13. The spores ofNC–4 are about 35 times more resistant to UV thanys–13 spores at 10% survival. Pyrimidine dimers were formed in UV-irradiated spores in both strains. No photoproducts other than pyrimidine dimers were detected. The formation of pyrimidine dimers in spores was about 2% in both strains at 800 J/m₂. In the germination of spores, the conversion of spores into swollen spores was not affected by UV in both strains, but the emergence of amoebae from the swollen spores was suppressed, which was more distinctive inys–13 spores than inNC–4 spores. The emerged amoebae from the UV-irradiatedNC–4 spores were viable, while those from theys–13 spores were inviable even when they succeeded in emergence.     

THE EFFECTS OF MAGNETIC FIELD ON GRAFT COPOLYMERIZATION OF METHYL METHACRYLATE ONTO POLYVINYL ALCOHOL IN THE PRESENCE OF BENZOPHENONE DURING UV IRRADIATION

The effects of magnetic field on the graft ratio and stereoregularity of grafts of PVA-g-MMA in the presence ofbenzophenone during UV irradiation are discussed. By means of IR, it was found that the graft ratio was increased with the increment of magnetic field strength. Furthermore, application of relative weak magnetic field of 0.4 Tesla had been shown to substantially enhance the stereo-regularity of graft copolymer. The maximum stereo-regularity appeared when the graft ratio approached to 85% with the magnetic field of 1.2 Tesla (T). The resistance to moisture and heat resistance of the grafted copolymer in the presence of magnetic field were also improved.     

VARIATION IN THE PHOTOCHEMICAL REACTIVITY OF THYMINE IN THE DNA OF B. SUBTILIS SPORES, VEGETATIVE CELLS AND SPORES GERMINATED IN CHLORAMPHENICOL*,†

Abstract— The chief photoproduct of thymine produced in u.v. irradiated (2537Å) vegetative cells of B. subtilis is the cyclobutane-type dimer while in spores very little of this dimer is produced (maximum yield 2·6 per cent of thymine) but a new photoproduct is produced in high yield (maximum of 28·4 per cent of thymine). This difference in photochemical response appears to be due, at least in part, to a difference in uydration of the DNA. The photochemistry of thymine in isolated DNA irradiated in solution is similar to that of DNA in irradiated vegetative cells, but differs markedly from that of isolated DNA irradiated dry. The yield of cyclobutane-type thymine dimer is much reduced in isolated DNA irradiated dry but a new photoproduct of thymine. is produced which is chromatographically similar to the spore photoproduct. The yield of this photoproduct, however, is never as great as that obtained in irradiated spores. The photochemistry of the DNA thymine of spores germinated in the presence of chloramphenicol is very similar to that of normal vegetative cells. Except for hydration, the physical state of the DNA is probably not otherwise altered by germination in the presence of chloramphenicol since DNA replication is prevented by the presence of chloramphenicol. These results are also consistent with the hypothesis that the unique photochemistry of spores is due, at least in part, to the hydration state of the DNA. The acid stability of the spore photoproduct is indicated by the fact that it is isolated from irradiated spores after hydrolysis in trifluoroacetic acid at 155°C for 60 min. It still contains the methyl group of thymine as judged by the fact that for a given dose of u.v. the same yield of photoproduct was obtained whether the spores were labeled with thymine-2–C-14 or -methyl-C-14. This photoproduct is stable to reirradiation (2537Å) in solution under condiditions where thymine dimers of the cyclobutane-type are completely converted back to monomeric thymine. On a column of molecular sieve material (Sephadex-G10), the spore photoproduct elutes in a region intermediate between the cyclobutanetype thymine dimers and monomeric thymine. Of the numerous compounds tested by paper chromatography, the spore photoproduct is most similar (but not identical) in several solvents to 5–hydroxyuracil and 5–hydroxymethyluracil. Our data do not allow us to decide if the product is a monomer or a dimer. Although the photochemistry of thymine in the DNA of spores differs markedly from that for vegetative cells, several lines of evidence make it seem doubtful that the enhanced resistance of spores to u.v. relative to that of vegetative cells can be explained solely on the basis of this difference in the photochemistry of DNA thymine.     

EFFECTS OF SOLAR AND ARTIFICIAL UV IRRADIATION ON MOTILITY AND PHOTOTAXIS IN THE FLAGELLATE, Euglena gracilis

The effect of solar irradiation on the percentage of motile cells, their average speed and their phototactic orientation to white actinic light was studied in the flagellate, Euglena gracilis. Unfiltered solar radiation in midsummer during mid-day at a location near Lisboa, Portugal, was found to impair motility within 2 h. This effect is exclusively due to the UV-B component of the radiation and not due to UV-A, visible light or a temperature increase. Likewise, phototactic orientation was drastically impaired. Reduction of the solar UV-B irradiation by insertion of an ozone-Hooded plexiglass cuvette partially reduced the inhibition and covering the cuvettes with glass prevented any decrease in motility and photoorientation. Similar results were found with artificial irradiation (Xe lamps). After inoculation the motility of the population follows an optimum curve (optimum at 8 days). Also, the UV-B effect on motility was smallest after about one week and increased for younger and older cultures.