EXPERIMENTAL CONTROL OF FLOWERING IN LEMNA. II. SOME EFFECTS OF MEDIUM COMPOSITION,CHELATING AGENTS AND HIGH TEMPERATURES ON FLOWERING IN L. PERPUSILLA 6746

Hillman , William S. (Yale U., New Haven, Conn.) Experimental control of flowering in Lemna. II. Some effects of medium composition, chelating agents and high temperatures on flowering in L. perpusilla 6746. Amer. Jour. Bot. 46(7): 489–495. Illus. 1959.—-L. perpusilla 6746 flowers as a short-day plant on Hutner's medium (containing ethylenediaminetetraacetic acid [EDTA]) at constant temperatures from 25 to 30°C., but eventually flowers also in old cultures under 16 or 24 hr. of light. This old-culture flowering is more pronounced in dilute medium. Flowering is rapid under both long and short days at constant temperatures from 25 to 28°C. in media not containing EDTA; the addition of 10^-5 M EDTA or of similar or higher concentrations of numerous other chelating agents suppresses flowering under long days but not under short (8 hr. light). This effect does not depend on promotion or inhibition of vegetative growth. At 29 to 30°C., a short-day requirement is manifested even in media permitting flowering under long days at the lower temperatures. Temperatures above 31°C. completely inhibit flowering under all conditions. Brief periods of high temperature given to plants under short-day conditions inhibit flowering when given during the dark period but not during the light period. The implications of these observations for the further study of flowering are discussed.     

Characterization of a photosynthetic mutant of Lemna lacking the cytochrome b6-f complex

A photosynthetic mutant of Lemna perpusilla (no. 1073) has been examined by spectrophotometric and immunoblotting techniques in order to localize the site of defect. In contrast to previous conclusions (Shahak, Y., Posner, H.B. and Avron, M. (1976) Plant Physiol. 57, 577-679), neither cytochrome f nor cytochrome b6 could be detected spectrophotometrically in the mutant. Furthermore, immunoblotting using antibodies specific for each of the four constituent subunits of the cytochrome b6-f complex demonstrate that the entire complex is absent in the mutant. The light-harvesting chlorophyll-protein complex of Photosystem II is present in similar amounts in wild-type and mutant Lemna. However, the total amount of plastoquinone-9 is reduced by approx. 65% in the mutant strain, while the photoreducible plastoquinone-9 pool is comparable in wild-type and mutant Lemna.     

X-Ray-Induced Chromosome Aberrations in Mouse Dictyate Oocytes. II. Fractionation and Dose Rate Effects

Split-dose experiments were done on maturing dictyate oocytes to determine if the magnitude of the first dose influenced the "rejoining time" of radiation-induced chromosomal lesions. A total dose of 400r was split into various combinations with varying fractionation intervals. The data derived from analyzing interchanges indicate that there is no difference in the rejoining time whether the first dose was 100, 200, or 300r. It thus appears that the radiation dose in the ranges studied does not significantly alter the rate of repair of the chromosomal lesions. This conclusion is contrary to that which has been propounded to explain the nonlinear dose curves obtained for specific locus mutations.

Chronic ⁶⁰Co γ-ray exposures were given to female mice over an 8-day period. The exposures were delivered during the period of peak sensitivity, i.e., 8–16 days prior to ovulation. The doses given were 117, 240, 348, and 483r. The aberration yields observed were dramatically lower than for comparable doses of acute X rays even when the RBE of γ rays compared with X rays is taken into account. The large drop in yields at the low dose rates is interpreted as resulting from a large two-track component in the acute curve, and as being independent of effects on repair systems.

     

Absence of a dose-fractionation effect on neoplastic transformation induced by fission-spectrum neutrons in C3H 10T1/2 cells

We have investigated the effect of fission-spectrum neutron dose fractionation on neoplastic transformation of exponentially growing C3H 10T1/2 cells. Total doses of 10.8, 27, 54, and 108 cGy were given in single doses or in five equal fractions delivered at 24-h intervals in the biological channel of the RSV-TAPIRO reactor at CRE-Casaccia. Both cell inactivation and neoplastic transformation were more effectively induced by fission neutrons than by 250-kVp X rays. No significant effect on cell survival or neoplastic transformation was observed with split doses compared to single doses of fission-spectrum neutrons. Neutron RBE values relative to X rays determined from data for survival and neoplastic transformation were comparable.     

EXPERIMENTAL CONTROL OF FLOWERING IN LEMNA I. GENERAL METHODS. PHOTOPERIODISM IN L. PEPUSILLA 6746

Hillman , William S. (Yale U., New Haven, Conn.) Experimental control of flowering in Lemna. I. General methods. Photoperiodism in L. perpusilla 6746. Amer. Jour. Bot. 46(6): 466–473. Illus. 1959.—Lemna perpusilla strain 6746 flowers as a typical short-day plant when grown aseptically in Hutner's medium (containing ethylenediaminetetraacetic acid, [EDTA]) at 26–28°C. A method is described for quantitatively assaying the degree of flowering in a culture. Maximal flowering takes place under photoperiods of 6–11 hr., and none under photoperiods exceeding 15 hr. The flower-promoting effects of long nights are inhibited by brief interruptions with red light, such interruptions being most effective in the middle of the dark period. A single long night will cause the subsequent production of flowering fronds, but vegetative growth in the culture is resumed after a time. Only frond primordia at a very early stage of development appear to be sensitive to induction. Quantitative flowering experiments lasting a week or less can easily be performed with this plant; it is ideally suited for studies of the effects of light, darkness, temperature, organic compounds and other factors under highly controlled conditions.     

Cell death (apoptosis) in mouse intestine after continuous irradiation with gamma rays and with beta rays from tritiated water

Apoptosis is a pattern of cell death involving nuclear pycnosis, cytoplasmic condensation, and karyorrhexis. Apoptosis induced by continuous irradiation with gamma rays (externally given by a 137Cs source) or with beta rays (from tritiated water injected ip) was quantified in the crypts of two portions of mouse bowel, the small intestine and descending colon. The time-course change in the incidence of apoptosis after each type of radiation could be explained on the basis of the innate circadian rhythm of the cells susceptible to apoptotic death and of the excretion of tritiated water (HTO) from the body. For 6-h continuous gamma irradiation at various dose rates (0.6-480 mGy/h) and for 6 h after injection of HTO of various radioactivities (0.15-150 GBq per kg body wt), the relationships between dose and incidence of apoptosis were obtained. Survival curves were then constructed from the curves for dose vs incidence of apoptosis. For the calculation of the absorbed dose from HTO, the water content both of the mouse body and of the cells was assumed to be 70%. One megabecquerel of HTO per mouse (i.e., 40 MBq/kg body wt) gave a dose rate of 0.131 mGy/h. The mean lethal doses (D0) were calculated for gamma rays and HTO, and relative biological effectiveness values of HTO relative to gamma rays were obtained. The D0 values for continuous irradiation with gamma rays were 210 mGy for small intestine and 380 mGy for descending colon, and the respective values for HTO were 130 and 280 mGy, indicating the high radiosensitivity of target cells for apoptotic death. The relative biological effectiveness of HTO relative to 137Cs gamma rays for cell killing in both the small intestine and the descending colon in the mouse was 1.4-2.1.     

Repopulation in murine skin after X-ray treatments with multiple fractions per day

The kinetics of repopulation of clonogens in skin after fractionated X-ray exposures was studied in a series of experiments using a top-up design. The feet of mice were exposed to small X-ray doses (1.5 or 2 Gy), given two or three times a day on consecutive days with a minimum interfraction interval of 8 h. A single top-up dose of d(4)-Be neutrons was then given at various intervals after the last X-ray fraction, typically on Days 1,4,8, 15, and 19. The acute skin reaction produced was scored an analyzed by both a standard 23-day averaging and a 7-day averaging procedure. Either method gave similar results and led to the same conclusions. The amount of top-up dose needed to produce a fixed skin reaction was used as a measure of the net effect of the X-ray treatments. This net effect is a result of the initial reduction in skin clonogens due to X rays, and their repopulation before the top-up dose was given. Repopulation was not detected during any of these courses of fractionated treatment, up to an overall time of at least 12 and possibly 16 days. On completion of X-ray schedules lasting 6-16 days, repopulation started 4 days later. In contrast, this delay lengthened to approximately 8 days for shorter overall treatment times of 3-4 days. Once repopulation started, it proceeded rapidly over 11 days so that by 15 days after the cessation of X rays, the skin was restored almost to its normal state with respect to radiosensitivity. The residual damage from Day 15 to Day 19 postirradiation was 3-13% of a full-effect level. The rate of repopulation can be expressed as a clonogen doubling time (Tclon), assuming that an average skin reaction of 1.5 is equivalent to a clonogen surviving fraction of 1.7 x 10(-5). Tclon varied inversely with the amount of initial damage inflicted by the X rays, with the shortest values (1-1.3 days) seen following X-ray doses that gave an initial damage level of 60-80% of full effect. These data are consistent with a hypothesis that damage is "sensed" only 10-12 days after the first X-ray fraction, which provides the stimulus for repopulation of the target cells in the basal layer, the keratinoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dose-response modeling of life shortening in a retrospective analysis of the combined data from the JANUS program at Argonne National Laboratory

Life shortening was investigated in both sexes of the B6CF1 (C57BL/6 x BALB/c) mouse exposed to fission neutrons and 60Co gamma rays. Three basic exposure patterns for both neutrons and gamma rays were compared: single exposures, 24 equal once-weekly exposures, and 60 equal once-weekly exposures. Ten different dose-response models were fitted to the data for animals exposed to neutrons. The response variable used for all dose-response modeling was mean after-survival. A simple linear model adequately described the response to neutrons for females and males at doses less than or equal to 80 cGy. At higher neutron dose levels a linear-quadratic equation was required to describe the life-shortening response. An effect of exposure pattern was observed prior to the detection of curvature in the dose response for neutrons and emerged as a potentially significant factor at neutron doses in the range of 40-60 cGy. Augmentation of neutron injury with dose protraction was observed in both sexes and began at doses as low as 60 cGy. The life-shortening response for all animals exposed to gamma rays (22-1918 cGy) was linear and inversely dependent upon the protraction period (1 day, 24 weeks, 60 weeks). Depending on the exposure pattern used for the gamma-ray baseline, relative biological effectiveness (RBE) values ranged from 6 to 43. Augmentation, because it occurred only at higher levels of neutron exposure, had no influence on the estimation of RBEm.     

Role of environmental factors on the reproducibility of Lemna test

Lemna minor is a species easy to collect and culture in laboratory, and can give rapid test results. However, in order to standardise toxicity tests using Lemna minor as test organism, it is important to find out what natural variability different populations might have. Five Lemna populations were used for comparison. It contained two standard cultures and three populations collected in natural habitats. Potassium dichromate was applied as test material. Lemna populations cultured under the same conditions showed different T(D) and LC50 values. There is an inverse relation between the sensitivity and T(D) of the strains. It is supposed that growth rate and sensitivity of Lemna populations depend on environmental factors characterising the habitat in which the given popluation originally lives.     

Investigations on Cytokinins. III. Cytokinin Activity in Lemna minor tRNA Hydrolysates

tRNA was extracted from Lemna minor, grown on a cytokinin free medium. Alkaline hydrolysates of the tRNA were active in three cytokinin bioassays: mobilization test, tissue culture and growth of Lemna cultures. Some observations on the growth of Lemna as a bioassay for cytokinins, are given.     

The effect of various aquatic bacteria on the growth and senescence of duckweed (Lemna minor)

Five different species of freshwater bacteria ( Pseudomonas sp., Vibrio sp., Klebsiella sp., Enterobacter sp., Serratia sp.) and a mixed natural population were used separately to inoculate cultures of axenic duckweed ( Lemna minor ). Inoculation with Vibrio sp. caused the final population density of Lemna plants to be significantly greater after 52 d than that of either axenic controls or Lemna inoculated with a mixed bacterial community. Inoculation with Pseudomonas sp. caused the final population density of Lemna to be significantly higher than with the mixed bacterial treatment. Inoculation of Lemna with Klebsiella sp., Enterobacter sp. or Serratia sp. resulted in higher plant populations compared with controls, but these differences were not statistically significant. The presence of a mixed community of bacteria did not significantly affect the final population density of Lemna compared with the controls. However, Lemna plants inoculated with a natural population of bacteria showed significantly higher levels of senescence compared with the other five treatments and the controls. None of the five single bacterial taxa used appeared to have any significant effect of the sensescence of duckweed.     

Studies on the Flowering Mechanism in Lemna

The dark reaction of the short day plant Lemna perpusilla was investigated. It was found that 3-phosphoglycerate and pyruvate (10^?6M) increased the flowering rate in the presence of nitrates. Pyruvate-2-¹⁴C was added to the culture solution during two hours of the dark reaction and ¹⁴C was incorporated into serine, aspartate and glutamate. It was postulated that pyruvate reacted with a nitrogen source forming an intermediate, possibly aspartate, which was further converted into serine. L. perpusilla failed to flower when the dark period was interrupted with red light and as a result endogenous serine accumulated in a high concentration. The dark reaction of L. perpusilla, in which serine was involved, required (1) oxygen, (2) ATP, (3) moderate temperature, and (4) an enzyme system.     

Reassessment of gamma doses from the atomic bombs in Hiroshima and Nagasaki

Reassessment of gamma doses from the atomic bombs in Hiroshima and Nagasaki has been carried out with thermoluminescent measurements of ceramic materials, such as bricks and decorative tiles, which were collected from buildings that remain as they were at the time of the explosions. The thermoluminescent measurements were performed using thermoluminescent dating techniques generally used in archaeology. Annual background dose rates from natural radionuclides in the ceramic materials and from environmental radiation including cosmic rays were determined with commercially available thermoluminescent detectors. A time-zero point at the original firing of the ceramic materials was estimated from the age of the buildings given in "the register book." Total background dose was evaluated by multiplying the period between the time-zero point and the time of measurement by the annual dose rate. The resultant gamma doses in Hiroshima and Nagasaki are given as a function of distance from ground zero and are compared with the DS86 (Dosimetry System 1986) and the T65D (Tentative 1965 Dose) gamma doses.     

Neoplastic transformation in vitro after exposure to low doses of mammographic-energy X rays: quantitative and mechanistic aspects

The induction of neoplastic transformation in vitro after exposure of HeLa x skin fibroblast hybrid cells to low doses of mammography-energy (28 kVp) X rays has been studied. The data indicate no evidence of an increase in transformation frequency over the range 0.05 to 22 cGy, and doses in the range 0.05 to 1.1 cGy may result in suppression of transformation frequencies to levels below that seen spontaneously. This finding is not consistent with a linear, no-threshold dose- response curve. The dose range at which possible suppression is evident includes doses typically experienced in mammographic examination of the human breast. Experiments are described that attempt to elucidate any possible role of bystander effects in modulating this low-dose radiation response. Not unexpectedly, inhibition of gap junction intercellular communication (GJIC) with the inhibitor lindane did not result in any significant alteration of transformation frequencies seen at doses of 0.27 or 5.4 cGy in these subconfluent cultures. Furthermore, no evidence of a bystander effect associated with factors secreted into the extracellular medium was seen in medium transfer experiments. Thus, in this system and under the experimental conditions used, bystander effects would not appear to be playing a major role in modulating the shape of the dose-response curve.     

Cholecystokinin-dependent selective inhibitory effect on 'minute rhythm' in the ovine small intestine

Cholecystokinin (CCK) can exert multiple actions on intestinal motility but its effect on the small-intestinal 'minute rhythm' (MR) is virtually unknown. Therefore, the electrical activity from the abomasal antrum, duodenal bulb, duodenum, jejunum and ileum was continuously recorded in six sheep before, during and after slow intravenous administration, of three doses each, of cholecystokinin-octapeptide (CCK-OP) and cerulein. In four of these sheep, two additional electrodes and the strain gauge force transducer were also inserted in the duodenum. Chronic experiments were performed in the fasted and non-fasted animals and saline or CCK peptides were injected during phases 1, 2a or 2b of the duodenal migrating myoelectric complex (MMC). The administration of both CCK peptides in various doses evoked an inhibitory effect mostly in the duodenal bulb, except for the lowest dose of cerulein. The effects of 20 times greater doses of CCK-OP than that of cerulein were more pronounced. The introduction of both CCK peptides during phase 1 of the MMC produced no marked or significant response. In non-fasted animals, the effects of both hormonal peptides, given during phase 2b of the MMC, were often stronger than those given during phase 2a, while in fasted animals the effects of CCK peptides, administered in the course of phases 2a and 2b of the MMC, were similar. Both higher doses of CCK peptides increased the number of spike bursts within the given MR pattern in the duodenum and decreased the incidence of MR mostly in the duodenal bulb. The inhibitory effects of both CCK peptides on the bulbar MR exhibited a dose-response character, though the lowest dose often evoked the slight stimulatory response. It is concluded that CCK principally exerts an inhibitory effect upon the MR in the duodenal bulb and modifies the MR in the duodenum by increasing the spike burst number in a given MR pattern. Both these actions of CCK peptides seem to be physiological. There is a positive relationship between the intensity of the refractory period and the demonstrated effect of CCK in the duodenum.     

Carbon Dioxide Output as an Index of Circadian Timing in Lemna Photoperiodism

Previous work on flowering suggested that photoperiodism in Lemna perpusilla 6746 involves an endogenous circadian "clock," but direct evidence requires study of an overt rhythm in the same plant. The CO(2) output rate of axenic cultures supplied with sucrose has been studied in a system using infrared analysis and monitoring four sets of cultures at once. Alternations of (1/4) to 21 hours of dim red light with darkness in 24-hour cycles can entrain the CO(2) output. In darkness following either continuous dim red light or entrainment to a 12(12) light (dark) schedule, the rate oscillates through two maxima and two minima, with a circadian periodicity, before apparently damping. In continuous red light, the rate is linear. The skeleton photoperiodic schedule (1/4)(5(1/2))(1/4)(18), with its two portions highly unequal, rapidly entrains the CO(2) output in a phase relationship which is the same irrespective of which dark period is given first. The schedules (1/4)(13)(1/4)(10(1/2)) and its inverse, however, with two portions more nearly equal in length, differ markedly from each other with respect to manner of entrainment, as they do in their effects on flowering. These and other results strongly support the concept that a circadian clock is an important component of photoperiodism, and they provide a new experimental system in which to study its action.     

METABOLIC INHIBITORS AND CHROMOSOME REJOINING

Beatty , Alvin V., and Jeanne W. Beatty . (Emory U., Atlanta, Ga.) Metabolic inhibitors and chromosome rejoining. Amer. Jour. Bot. 46(5): 317–323. 1959.—X ray-induced chromosomal aberrations of the dicentric and ring types in the first microspore division in Tradescantia paludosa were used to study the effects of temperature and chemical substances acting as metabolic modifiers on rejoining under both aerobic and anaerobic conditions. A total dose of 400 r of x rays was administered at 50 r/min. at temperatures ranging from 0.3°C. to 45°C. In the aerobic temperature experiments, 5% oxygen was used in either helium or carbon monoxide and in the anaerobic ones helium was used. In 5% oxygen in helium, the highest number of aberrations was produced at the lowest temperature. When the amount of oxygen in solution was made constant at all temperatures by adjusting the positive pressure of gas in experiments carried out above zero degrees, there was a temperature effect at 0.3°C. and 10°C., but no effect from 20°C. to 45°C. Temperature was believed not to influence chromosome breakage but only rejoining, through its influence on oxidative metabolism, thereby removing the source of energy. In the anaerobic temperature experiments, a low aberration yield of 0.14 occurred at 0.3°C., while a high of 0.66 was recorded at 40°C. Cytochrome oxidase was found not to be involved in the energy supply for rejoining in anaerobic experiments, since exposures in alkaline pyrogallate-washed carbon monoxide gas gave the same results as helium, indicating that both gases only provide anoxic conditions. Under anoxic conditions, pretreatment in M/1000 sodium iodoacetate or M/10,000 p-chloromercuribenzoate solutions had no effect on non-irradiated plants thus treated, but increased the average number of chromosome aberrations per cell produced by x rays at least 2-fold over the irradiated controls. The effect of these 2 salts, as used in these experiments, is believed to have taken place through their inactivation of triosephosphate dehydrogenase, thus inhibiting glycolysis at an early stage before any energy was released.     

Survival of cultured mammalian cells exposed to ultrasound

Summary The colony-forming ability of cultured mammalian cells exposed to monochromatic ultrasonic vibrations of different frequencies (0.1, 0.5, 1.0, 2.0, 3.3 MHz) was studied. The combined effect of x rays and 1.0-MHz ultrasonic waves on the survival of M3-1 cells was also investigated. Split-dose experiments showed that cells exposed to ultrasound are sensitized to a subsequent exposure. Almost twice as many cells survive a given ultrasonic dose when exposed in the M phase as when exposed in the S phase. A small amount of synergism between ultrasound and x rays has been observed. The extent of synergism depends on the experimental factors, and may be due to an interaction between nuclear damage caused by x rays and the damage to the cell membrane caused by ultrasound.Research supported by the U.S. Atomic Energy Commission and the National Aeronautics and Space Administration     

Does weak local gamma irradiation modify the effect of CDDP on mouse mastocytoma?

The possibility that an irradiation with small doses of gamma rays given shortly before might enhance the cure effect of cis-diammine-dichloroplatinum (II) (CDDP) was tested in tumor bearing mice. Tumor cells of FMA3, a cloned cell line of murine mastocytoma, were transplanted in the muscle of a hind leg in one experiment and in the abdominal cavity in another experiment. In both cases, a single injection of CDDP at 4 mg/kg was effective to lengthen the mean survival time of mice. gamma rays at the dose of 1, 2 and 4 Gy given 3 hours before did not modify the effect of CDDP.     

Induced mutations in foxtail millet (Setaria italica Beauv.)

Summary Viable mutations for ear characters in two cultures of foxtail millet (Setaria italica Beauv.) were induced by individual and combined treatments of gamma rays, EMS (ethyl methanesulphonate), and dES (diethylsulphate). Four doses of gamma rays (10Kr to 40Kr), and four durations (6hrs to 24hrs) each of EMS (0.1 %) and dES (0.1 %) were used. In the combined treatments, only two durations (6hrs, 12hrs) of the chemical mutagens were used following each of the four doses of gamma rays.The mutation frequencies were recorded as mutants per 100 M₂ plants and these were found to be higher in MU-1 than in MU-2 in all the treatments except dES. The frequencies also increased with increase in dose or duration of treatments and were found to be much higher in the combined treatments than in the individual treatments. In several combined treatments, a synergistic effect was observed in culture MU-1, the degree of synergism ranging from 1.01 to 1.62. Thus there was a differential response by the two cultures to the mutagenic treatments. The mutation spectrum was also found to be wider in culture MU-1, where 11 different kinds of mutants were recorded compared with only eight kinds of mutants recorded in MU-2.