Sensitization of three strains of chlorococcal algae for UV- effects by 5- bromodeoxyuridine

The sensitization of chlorococcal algae by 5-BdU for the purpose of UV-light mutagenesis was studied. The results obtained were compared with our earlier findings on the sensitization of the same algal strains by 5-BU. No shielding effect of the 5-BdU molecules against UV-light was observed. Probably, the uptake of them from the liquid medium did not result in such excess as compared with the treatment by 5-BU, even if the cells were long enough (24 h) exposed to the concentration of 5-BdU. Likewise, neither stimulating nor inhibiting growth effects on the growing cell colonies were observed after treatment with 5-BdU. The sensitization of the algal cells for UV-light effects was effective in all the experiments. An increased damage of the algal cells by UV-light after sensitization was proved in all the parameters recorded. The frequencies of permanent changes of the cells or their colonies were also increased, but their spectrum did not change significantly. A suitable combination of the 5-BdU sensitization of the cells before their influencing by UV-light and the induction of their repair mechanisms by visible light may decrease the frequencies of the lethal or sublethal damage and increase the frequencies of the useful permanent changes in the characteristics of the chlorococcal algae. The results obtained are discussed from the viewpoint of the regulated mutation process in the breeding of algae.     

Radiation-induced mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

A number of genetic systems are described which in yeast may be used to monitor the induction of chromosome aneuploidy during both mitotic and meiotic cell division. Using these systems we have been able to demonstrate the induction of both monosomic and trisomic cells in mitotically dividing cells and disomic spores in meiotically dividing cells after both UV light and X-ray exposure. The frequency of UV-light-induced monosomic colonies were reduced by post-treatment with photoreactivity light and both UV-light- and X-ray-induced monosomic colonies were reduced by liquid holding post-treatment under non-nutrient conditions. Both responses indicate an involvement of DNA-repair mechanisms in the removal of lesions which may lead to monosomy in yeast. This was further confirmed by the response of an excision-defective yeast strain which showed considerably increased sensitivity to the induction of monosomic colonies by UV-light treatment at low doses. Yeast cultures irradiated at different stages of growth showed variation in their responses to both UV-light and X-rays, cells at the exponential phase of growth show maximum sensitivity to the induction of monosomic colonies at low doses whereas stationary phase cultures showed maximum induction of monosomic colonies at high does. The frequencies of X-ray-induced chromosome aneuploidy during meiosis leading to the production of disomic spores was shown to be dependent upon the stage of meiosis at which the yeast cells were exposed to radiation. Cells which had proceeded beyond the DNA synthetic stage of meiosis were shown to produce disomic spores at considerably lower radiation doses than those cells which had only recently been inoculated into sporulation medium. The results obtained suggest that the yeast sustem may be suitable for the study of sensitivities of the various stages of meiotic cell division to the induction of chromosome aneuploidy after radiation exposure.     

Responses of cell populations of three chlorococcal algae to the action of streptomycin

Time doses of a single concentration of streptomycin and its concentration doses acting for the same time period in a liquid medium had different effects on three strains of chlorococcal algae. This concerned both the physiological responses and permanent changes in the characteristics of cell colonies growing from treated cells. Significant differences were recorded in: the number of autospores produced during the first division of the treated cells on the surface of a solid medium, the length of the lag phase, the growth rate of the diameter of cell colonies, and the survival of the treated cells. The permanent changes in the characteristics of the growing colonies were very different in the individual algal strains in quality and frequency. Physiological and the mutation effects were compared and discussed.     

Effects of ultraviolet irradiation on structural and functional condition of T- and B-lymphocytes of human blood

The influence of ultraviolet (UV) radiation on structural and functional condition of membranes T- and B-lymphocytes of human blood is investigated. It is shown, that intergral flow of UV-light (240-390 nm) in doses 151-1359 J/m2 leads to the increase of the expression of some HLA-antigen (DR1, DR2, DR5, DR7) and Fc-receptors on membranes of B-lymphocytes. The level of HLA-antigen was increased by 20-500% from the initial state after exposure to different doses. The growth of the expression of Fc-receprots was registered in 80% of the donors in the range from 20 up to 540%. The minimum dose of UV-light (151 J/m2) caused the maximum effect. The reduction of the level of Fc-receptors by 10-90% relatively to the native condition was registered for T-cells in 80% of the donors. Groups of the donors were revealed, T-lymphocytes of which differed by the sensitivity to UV-radiation. The effect of "strengthening--weakening", resulting to the reduction of the initially high parameters and to increase in the initially low ones was found.     

Growth of Streptococcal Protoplasts and L-Colonies on Membrane Filters

Membrane filters (Millipore Corp.; pore sizes 1.2 to 0.22 mum) were placed on the surface of L-phase growth medium solidified with agar. The filter and the surrounding medium were inoculated with either protoplasts or stable broth-grown L-phase variants obtained from Streptococcus faecium strain F24. The L-phase inoculum gave rise to viable L-colonies on the filters and on the medium, whereas protoplasts gave colony formation only on the medium. However, when the Millipore filters were covered by a layer of solid L-phase medium, 75 mum or greater in depth, before inoculation with protoplasts, colony formation resulted but with atypical morphology. In contrast, inoculation of protoplasts on Nuclepore and Sartorius membrane filters did give rise to L-colonies on the surface and underneath the filters after 2 days of incubation at 37 C. Submicroscopic, viable L-phase elements produced during colony formation were capable of passing through membrane filters with pore channels as small as 0.22 mum; these elements required transfer from underneath the filters to fresh agar medium in order to develop into L-phase colonies. Membrane filters were also placed on the surface of L-phase growth medium solidified with gelatin. Inoculation of the filters and surrounding medium with a lysozyme-prepared protoplast suspension gave rise to streptococci on the surface of the filters and on the medium. However, inoculation with the stable broth-grown L-phase variants gave rise to atypical colonies on the medium and only small patches of abortive growth on the filters.     

Effect of various peptides on the growth of Mycobacterium tuberculosis

Ten oligopeptides containing asparagine, glutamic acid, leucine or alanine on growth of Bacillus tuberculosis were tested. The experiments were performed on AS medium free of peptones. Bacterial suspensions were inoculated and the number of colonies and rapidity of bacilli growth under an influence of peptides tested was compared. Out of peptides studied and their different combinations the best turned to be combination of 0.01% glutathione +0.002% Gly-Asn + 0.0033% Leu-Gly. This combination allowed to appear on average 46% colonies more than on medium without peptides and first growth of tubercle bacilli was seen on average 3.2 days earlier than on medium free of peptides. Addition to the medium containing three above listed peptides of 0.1% of Bacto Tryptone (Difco) caused an increase of 127% of colony number of tubercle bacilli and their growth appeared 1.7 day earlier as compared to growth on medium containing these three peptides.     

Mutation of the Methane Oxidizing Bacterium, Methylococcus capsulatus

S ummary : Spontaneous mutants of Methylococcus capsulatus resistant to antibiotics, amino acid analogues and other compounds were obtained at frequencies similar to those found in other bacteria. Attempts to increase these frequencies with the mutagens N-methyl-N'-nitro-N-nitrosoguanidine, N-nitroso-N-methyl urethane, ethyl methane-sulphonate and UV were unsuccessful. Using these mutagens, only one auxotrophic mutant was isolated from 11,082 colonies examined. The growth characteristics of this p -aminobenzoic acid requiring mutant are described.     

Factors affecting the quantitation of dose-response curves for mutation induction in V79 Chinese hamster cells after exposure to chemical and physical mutagens.

Using four common mutagens, ethyl methanesulphonate (EMS), methyl methanesulphonate (mms), uv, and X-irradiation, the relationship between dose of mutagen, cellular lethality and frequency of 8-azaguanine resistant colonies in V79 Chinese hamster cells has been examined. Several factors affecting the recovery of mutants including inter and intra-clone metabolic co-operation have been quantitated and their influence on survival response curves examined. Induced mutant frequencies were assayed by two methods in situ, and after replating. After exposure to X-rays, MMS and UV a significantly higher frequency of mutants was observed in replated experiments as compared with the in situ situation, at all survival levels assayed. With EMS, an increment on replating was observed only at high survival levels. The replating data suggest that two types of azgr colonies are produced, i.e. those which contain only azgr cells and those which, due to damage segregation, contain a mixture of azgr and azg8 cells. These mixed colonies appear to be lost by metabolic co-operation when mutation frequencies are assayed in silu. The proportion of mixed to homogeneous colonies differs with different mutagens. Taking into account such factors, EMS and UV irradiation were similarly mutagenic at a given survival level, but at equitoxic doses, fewer mutants were recovered after exposure of V79 cells to MMS and X-rays.     

Light regime effect on the growth rate variability ofScenedesmus quadricauda (Turp.) Bréb. Clones originating from cells treated by a mutagen when grown on the surface of a solid medium

The light regime effect on the growth rate variability of clones of one chlorococcal alga strain growing on the surface of a solid medium was studied from the point of view of the clone selections manifesting the desired grade of this complex character. Several combinations of light regimes used for the cultivation of coenobia treated by UV-light and untreated control on two types of solid media proved the significance of manifestation degree changes of this character due to the given light conditions. The sizes of the alga colonies after a certain growth time on the surface of solid medium were evaluated statistically and the values obtained indicated the degree of growth rate manifested by the clones of a given population. The \(\bar x\) and s_x values of their growth rate variability were considered as the indicators facilitating the choice of selec-tion type and screening level. This paper is a partial contribution to the solution of the problems concerning the primary selection of alga clones on the surface of solid media.     

The use of the Salmonella/microsomal assay to determine mutagenicity in paired chemical mixtures.

The mutagenicities of two sets of chemicals acting singly and in pairwise combinations were determined by use of the Salmonella/microsomal assay. The first set consisted of the promutagens of benzo(a)pyrene and benzo(rst)pentaphene. The second set contained the direct-acting mutagens methyl-nitro-nitroso-guanidine and ethyl methane sulfonate. In the tests with the promutagens, the quantities of S-9 mix were varied over the range of 0.05 ml to 1.0 ml with increasing quantities of each chemical. The mutagenic responses or production of revertant colonies of the promutagens, acting singly and in pairwise combinations failed to show an additive effect. Excess quantities of S-9 mix appeared to inhibit partially or totally the mutagenic activity of each chemical, although for each particular dose there was an optimal quantity of S-9 mix to induce maximum activity. However, the direct-acting mutagens produced, individually, almost linear dose responses with increasing concentrations. In pairwise combinations, these chemicals also showed linear responses that closely approximated the theoretical additivity indicating that the mutagenicity of the mixtures was the sum of the activities of each component.     

Level of receptor complex molecule expression of human blood T-lymphocytes under conditions of their UV-irradiation

The influence of a wide range of doses of UV-light (240-390 nm) on the expression level of receptor complex molecules (CD3, CD4, CD8 markers) on the membrane surface of human blood T-lymphocytes has been studied using flow cytometry and enzyme linked immunosorbent assay. UV-light at small and medium doses (15 1, 453 and 906 J/m2) has been established to have a unidirectional (activating) effect on the expression level of receptor complex molecules, and at a high dose of 1359 J/m2 it can either increase (CD4 and CD8 markers) or reduce (CD3 complexes) the quantity of the analyzed molecules on the membrane surface of T-lymphocytes.     

Isolation Method for Bacterial Isolates Capable of Growth on p-Chlorobiphenyl

A method is reported for screening for p-chlorobiphenyl (pCB)-degrading bacteria from various environments. A solid medium was inoculated with the sample to be analyzed, colonies were allowed to develop, and the plates were then sprayed with a pCB solution in ether. The positive colonies were recorded as those surrounded with a clear zone in the film of pCB. That these colonies were able to degrade pCB was shown by their ability to grow on pCB in liquid medium with concomitant disappearance of the substrate and by the appearance of colored compounds in cultures grown on pCB.     

金鱼藻(Ceratophyllum demersum Kom.)对藻类的生化干预作用

室内试验表明金鱼藻对普通小球藻(Chlorella vulgaris Beij.)和斜生栅藻(Scenedesmusobliquus(Turp.)Kutz.)的生长有明显的生化干预作用。本研究表明,金鱼藻和藻类共同培养,用培养过金鱼藻的培养液直接培养藻类,或用此培养液中提取的生物碱作藻类抑制实验,藻类的生长均受到抑制。等量的金鱼藻经煮沸后,对藻类的抑制作用高于原液。金鱼藻植株及其培养液中生物碱的含量与培养条件和时间有关。     

The effect of liquid holding on chemical induced lethality and mitotic gene conversion in Saccharomyces cerevisiae

Summary Mitotic gene conversion was induced with a variety of chemical mutagens in a double heteroallelic strain of Saccharomyces cerevisiae. Cells were treated with various mutagens and plated immediately onto selective and nonselective growth medium or else they were subject before plating to liquid holding in buffer for various lengths of time. In respiratory competent cells liquid holding caused a decrease in lethality and in conversion frequencies. Respiratory deficient cells, unable to use a non-fermentable substrate as an energy source, behaved different. Untreated cells started to die in buffer after two days of storage, and moreover, there was a considerable increase in potential convertants i.e. cells giving rise to gene convertants when plated on selective growth media. Respiratory deficient cells treated with various chemical mutagens were still more sensitive to liquid holding. After low, sublethal doses cells started to die after one day of liquid holding already and when plated on media selective for convertants, showed an increasing frequency of gene convertants. Addition of very low concentrations of glucose to the liquid holding buffer post-poned the lethal and convertogenic effects. Higher concentrations of glucose completely abolished sensitivity to liquid holding-induced lethality and genetic alterations. The results are interpreted to mean that in respiratory deficient cells no repair activities are possible to an accumulation of spontaneous lethal damage and genetic alterations which are expressed as gene conversion when an energy source becomes available. Such a repairless condition causes an increased sensitivity to genetically active agents, and provides a useful system to detect genetic effects of slowly reacting agents.     

Regulation of biosynthesis of secondary metabolites

The biosynthetic activity of mutants with altered morphological and biochemical characteristics, obtained from two strains ofStreptomyces aureofaciens by means of physical and chemical mutagens, was studied. The majority of mutants formed pigments different from pigments of the parent strains, which did not usually give fluorescence in UV-light and, in addition, differed as to the solubility in organic solvents. The production of further secondary metabolites was investigated chromatographically in the extracts from mycelium and in the fermentation fluid of submerged cultures. According to the results of chromatographical analysis, the obtained mutants can be divided into 12 metabolic types. In most of them the production of substances was found which are different both mutually and from the metabolites of parent strains in physical and chemical properties. A direct correlation was observed between the character of colonies and biosynthetic properties of mutants.     

Effects of antibiotics on UV-stimulated tube growth ofPinus silvestris pollen

Summary Studies were made to investigate the effects of different antibiotics on unirradiated pollen and on pollen with enhanced tube growth, stimulated by low doses of UV-light. The antibiotics mitomycin, chloramphenicol, tetracyclin, penicillin, nystatin and carbony-cyanid phenylhydrazon were not able to suppress tube growth stimulation of pine pollen. The data obtained are discussed in view of the stimulation mechanism of low doses of UV-light.     

Effect of formic acid formulations on honey bee (Hymenoptera: Apidae) colonies and influence of colony and ambient conditions on formic acid concentration in the hive

The interaction between the effects of varroa, Varroa destructor Anderson & Trueman, and formic acid treatments on colonies of honey bees, Apis mellifera L., were examined in two field experiments. In experiment 1, colonies with low varroa levels were exposed to two different slow-release formulations and compared with untreated colonies. In experiment 2, colonies inoculated with varroa and uninoculated colonies were exposed to a slow-release formulation, a pour-on formulation, or were left untreated. The effects of treatments, hive temperature, and hive relative humidity on formic acid concentration in hive air also were examined. Slow-release formic acid application improved colony development in colonies that had been inoculated with varroa. However, in uninoculated colonies where the mean abundance of varroa was low, slow-release formic acid application suppressed colony development. The pour-on application did not have a negative impact on worker population growth in uninoculated colonies, but also it was not as effective as the slow-release treatment in improving population growth in varroa-inoculated colonies. Equivalent volumes of acid applied in pour-on and slow-release formulations provided the same cumulative dose in hive air but differed in the daily pattern of formic acid release. Colonies that were not inoculated with varroa had higher concentrations of formic acid in hive air than colonies that were inoculated with varroa on three of the five pour-on application dates. The data suggest that reductions in worker population and/or activity caused by varroa can interact with ambient conditions to affect the volatilization or sorption of formic acid in the hive.     

The growth of potential food poisoning organisms on chicken and pork muscle surfaces

Muscle surfaces of pork were inoculated with a mixture of Yersinia enterocolitica and Staphylococcus aureus , and chicken muscle with Campylobacter jejuni or a mixture of Salmonella typhimurium and Staph. aureus . The surface growth at 20°C was followed microscopically. Organisms grew as discrete colonies bound together by a glycocalyx which differed between bacterial species. On prolonged incubation colonies spread peripherally and tended to coalesce, while still retaining their colony structure. Staphlycoccus aureus colonies were very small and remained so. The glycocalyx was considered critical in maintaining the dense populations of bacteria on the meat surfaces.     

Cloning and characterization of endosymbiotic algae isolated fromParamecium bursaria

Summary The green parameciumParamecium bursaria has many endosymbiotic algae in its cytoplasm. Here, we cloned and characterized endosymbiotic algae fromP. bursaria and examined in detail the interaction between the cloned algae and algae-free paramecia. Homogenates ofP. bursaria were cultured on agar plates containing various kinds of media to establish clones of the endosymbiotic algae. Many algal colonies were obtained from poorly nutritious medium (CA medium) after one month in culture. Algae were picked up from these colonies and inoculations were repeated 9 times on agar plates containing CA medium. On enriched media including bacto-peptone, glucose, proteose-peptone and/or yeast extract, however, bacteria and mold grew rapidly and no algal colonies were formed. When the cloned algae were cultured in liquid CA medium, they grew faster than on agar plates and the numbers stayed constant at 1 × 10⁷ algae/ml after 7 days in culture. They revealed high infectivity to algae-free paramecia, and an incubation period of 24 h and at least 1 × 10³ algae/paramecium were required to achieve successful infection (80–90%). The growth and infection rate did not change through 74 repeated inoculations of algae in liquid CA medium. Optical microscopic observations revealed marked morphological similarity between endosymbiotic algae and free-livingChlorella, but the latter showed no infectivity to algae-free paramecia. The cloned endosymbiotic algae presented here will provide an excellent opportunity to examine the mechanism of symbiont-host interaction.     

The growth of potential food poisoning organisms on chicken and pork muscle surfaces

Muscle surfaces of pork were inoculated with a mixture of Yersinia enterocolitica and Staphylococcus aureus, and chicken muscle with Campylobacter jejuni or a mixture of Salmonella typhimurium and Staph. aureus. The surface growth at 20 degrees C was followed microscopically. Organisms grew as discrete colonies bound together by a glycocalyx which differed between bacterial species. On prolonged incubation colonies spread peripherally and tended to coalesce, while still retaining their colony structure. Staphylococcus aureus colonies were very small and remained so. The glycocalyx was considered critical in maintaining the dense populations of bacteria on the meat surfaces.