Morphological,growth, and chromosomal changes in bovine pancreatic duct epithelial cells exposed toN-methyl-N′-nitro-N-nitrosoguanidine

Summary Epithelial cells cultured from bovine pancreatic ducts were given a single treatment ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Multinucleated cells and giant cells were observed more frequently in carcinogen-treated cultures than in controls. The MNNG-treated cultures also contained a sizeable population of small, dense cells that were not observed in control cultures. At the concentration of 1.0 μg/ml, MNNG caused an initial depression in the growth rate of the cells followed by growth stimulation for several weeks. The MNNG produced chromosomal damage in the cells as indicated by the observation that a substantial proportion of carcinogen-treated cells were heteroploid and contained a high frequency of metacentric and submetacentric chromosomes and a dicentric marker chromosome. The MNNG treated and control cultures did not acquire the ability to grow in soft agar or to produce tumors after transplantation into athymic, nude mice. This work was supported in part by Public Health Service Contract and Interagency Agreement Y01-CP60204 from the Division of Cancer Cause and Prevention, National Cancer Institute.     

Infection of procaryotic and eucaryotic microorganisms withMetallogenium

To elucidate the biological characteristics ofMetallogenium, a study was undertaken into the effect of infection of such organisms unable to oxidize manganese as fungi, yeasts, and bacteria with an ultrafiltrate of this organism on the subsequent behavior of the resulting binary cultures. The infection of microorganism cultures is accompanied by (a) acquisition by the binary cultures of a persistent capacity to oxidize manganese, (b) evolution of the characteristic structures ofMetallogenium (c) inhibition of the growth of the inoculated cultures; the inhibition manifests itself by fungi losing their capacity for spore formation and pigmentation, the upsetting of cell division processes, and lysis of the cells of the infected cultures all the way to death. When a heated ultrafiltrate ofMetallogenium was used to infect microorganism cultures, no signs of infection were detected.Metallogenium may be hosted by an extremely broad range of microorganisms that are in no way allied to one another. The experimental results suggest thatMetallogenium is an organism capable of parasitizing lower eucaryotic and procaryotic microorganisms. Analysis of 134 strains of fungi isolated from freshwater bodies is indicative of possible parasitism ofMetallogenium on microorganisms in their natural habitats.     

Effects of cadmium and copper on the ultrastructure ofAnkistrodesmus braunii andAnabaena 7120

The effects of brief exposure to, or growth in the presence of, lethal and sublethal concentrations of Cu(NO)₂ and Cd(NO₃) on the ultrastructure of the blue-green algaAnabaena 7120 and the green algaAnkistrodesmus braunii were studied. Exposure to increasing amount of both metal ions led to the appearance of larger proportions of electron-dense cells whose organelles were less well defined than those of untreated cells. Metal-treated cells ofAnabaena 7120 became distorted. Some had a corrugated appearance. Others lysed, leaving a much larger proportion of heterocysts. Such heterocysts were often empty or had a curious collapsed appearance. Growth ofA. braunii in the presence of 10^–4 M Cu(NO₂)₂ produced substantial numbers of multinucleate giant cells with thick walls; such cells result from repeated mitotic division without subsequent cytokinesis. The giant cells contained centrioles, structures not as yet found in normal cells of the genusAnkistrodesmus. Some nuclei of giant, but not of normal, cells contained deep indentations that appeared as holes in cross section. Some giant cells also contained triple parallel strands of endoplasmic reticulum which extended across much of the cell, connecting to the nuclear envelope. Some ultrastructural changes were also noted in algal cells grown over sediment containing Cu or Cd, but these were generally less severe than those occurring when metal ions were added directly to the algal cultures.     

An immunofluorescent technique for rapid control of the purity of yeast cultures

Summary A rapid technique for control of purity of cultures ofCandida utilis can be based on fluorescent labeling of cells using an antiserum raised in rabbits and rendered specific by absorption with cross-reacting strains. Microscopical observation under mixed ultraviolet/visible illumination permits detection of one non-fluorescent contaminant cell among 10³ fluorescent cells. Cells of all ages of allC.utilis strains tested reacted with the antiserum. Seven cross-reacting species were found among the 53 species from 15 genera of yeast tested.     

Binding and activity ofBacillus thuringiensis delta-endotoxin to invertebrate cells

Fluorescein isothiocyanate was used as a label to detect delta-endotoxin ofBacillus thuringiensis subsp.thuringiensis andisraelensis in binding studies with different in vitro cell systems. Protoxin of the subspeciesthuringiensis could be labelled directly whereas the activated toxin had to be traced indirectly with labelled antibodies. Both protoxin and activated toxin bound to primary midgut cell cultures ofPieris brassicae larvae as well as to cells of an established culture ofDrosophila melanogaster. No binding with either toxin form could be observed with hemocytes ofP. brassicae. Biological activity as shown by the trypan blue viability assay was obtained only with the activated toxin against the midgut cells. Toxin of the subspeciesisraelensis reacted very unspecifically. Binding followed by rapid destruction was obtained with all the tested cultures.Abbreviation FITC fluorescein isothiocyanate     

The fate ofsepia in small populations ofDrosophila melanogaster

Asepia gene found inD. melanogaster collected in North Carolina, and wildtype flies from North Carolina, Bogotá, Barcelona, and California were used to strt 120 cultures that were maintained by mass transfers of adults every third week for more than a year. The frequency ofsepia was determined in these cultures at the termination of the experiment. Thesepia gene was present in considerable frequency (16%–65%) in all backgrounds except one; in cultures involving wildtype chromosomes from North Carolina, it was virtually eliminated. Each of the wildtype backgrounds exhibited a characteristic final frequency ofsepia, suggesting that they had reached at least quasi-stable equilibria. Although it is likely that the retention ofsepia depended upon the superiority of flies heterozygous for this mutant, the technique does not reveal whethersepia itself was involved in the apparent heterosis.The work reported here was done under Contract No. AT-(30-1)-2139, U. S. Atomic Energy Commission.     

The ultrastructure of the eyes of<Emphasis Type="Italic"> Paravortex karlingi</Emphasis> (Plathelminthes,Rhabdocoela)

Both eyes of Paravortex karlingi belong to the rhabdomeric type. Each eye consists of a single pigment cup cell and three sensory cells. Modified mitochondria lie in three protrusions of the cup cell, and several of these mitochondria form giant mitochondrial derivates. The centres of the derivates include electron-dense substances. These modified structures may have lenticular functions. Furthermore, it is shown that giant mitochondrial derivates develop by fusion of smaller ones. Embryos have many small mitochondria in the developing eyes whereas the adult possesses less numerous, but giant forms. This circumstance leads to the assumption that all such lenticular structures within the rhabdocoels result from identical processes.     

Ricerche Sulla Cultura Sommersa di Cellule Singole di Piante Superiori

Abstract

Research on submerged culture of single cells of higher plants. — The author describes a method which allows to obtain submerged cultures of single cells of Phaseolus vulgaris and Nicotiana tabacum. The medium composition in macroelements in the culture on agar appears to effect to a great extent the ability of tissues to dissociate into single cells in the subsequent liquid culture. In this respect Heller's solution results to be more suitable than Gautheret's and Hildebrandt and Ri-ker's.

Cells are grown at 24 [ddot]C in 300 ml flasks containing 60 ml of broth on a rotary shaker at 220 rpm.

To prevent contaminations some antibacterial agents were added to cultures of Phaseolus vulgaris. Among these Penicillin and Neomycin were not tossic at 20 and 5 ppm concentrations respectively.

The presence of septa, which are observed also in largely vacuolate cells, seems to confirm the ability of single cells to divide.

The optimum 2,4-D concentration for growth decreases from 6 × 10^-8 to 6 × 10^-8 during successive liquid cultures, each of them being inoculated with on amount of the previous one. This fact, showing the adaptation of liquid cultures to decreasing concentrations of the growth hormone, is in agreement with previous observations in solid cultures by several authors.     

Physiological comparisons of transformed (crown gall) and nontransformedVinca rosea L. Cells

Summary In vitro growth rates of transformed (crown gall) and nontransformed cultures ofVinca rosea L. were greater at 32°C than at 25°C. The growth of transformed cells was significantly inhibited by kanamycin, neomycin, and chloramphenicol but not by cycloheximide. Nontransformed cells were inhibited by all four antibiotics., The relative growth rates of transformed cultures induced by four different strains, ofAgrobacterium tumefaciens did not correspond to the relative rates of tumor weight increase observed in vivo nor with the relative weights of tumor tissue in, plants 8 weeks after inoculation with the corresponding bacterial strains.     

Anaerobic biodegradation ofPara-cresol under three reducing conditions

The anaerobic degradation ofp-cresol was studied with one sediment source under three reducing conditions—denitrifying, sulfidogenic, and methanogenic. Loss ofp-cresol (1 mM) in all the anaerobic systems took initially 3 to 4 weeks. In acclimated culturesp-cresol was degraded in less than a week.p-Cresol was completely metabolized under denitrifying, sulfidogenic, and methanogenic conditions, with formation of nitrogen gas, loss of sulfate, and formation of methane and carbon dioxide, respectively.p-Cresol metabolism proceeded throughp-hydroxybenzal-dehyde andp-hydroxybenzoate under denitrifying and methanogenic conditions. These compounds were rapidly degraded in cultures acclimated top-cresol under all three reducing conditions. These results suggest that the initial pathway ofp-cresol degradation is the same under denitryfying, sulfidogenic, and methanogenic conditions and proceeds via oxidation of the methyl substituent top-hydroxybenzaldehyde andp-hydroxybenzoate. The initial rate ofp-hydroxybenzaldehyde degradation was high in both the unacclimated cultures and in the cultures acclimated top-cresol, suggesting that this step is nonspecific. Benzoate was additionally detected as a metabolite followingp-hydroxybenzoate in the methanogenic cultures, but not in the denitrifying or sulfidogenic cultures. The degradation pathway therefore may diverge afterp-hydroxybenzoate formation depending on which electron acceptor is available.     

Preliminary study of taxonomy ofAzotobacter andAzomonas by using rocket line immunoelectrophoresis

Rocket line immunoelectrophoresis was used to study the taxonomy ofAzotobacter andAzomonas assessed by reaction with antiserum to the AVO₂ strain ofAzotobacter. Forty-five cultures, comprising seventeen species in five genera, showed that antigen “β”, like high-titer somatic agglutination, was restricted to all 11 strains ofAzotobacter vinelandii and to one strain which has been namedAzotobacter macrocytogenes (10EM). A thermoresistant antigen (“γ”) was found to be shared by all strains and species ofAzotobacter andAzomonas investigated.     

A natural focus ofHistoplasma capsulatum var.duboisii is a bat cave

The natural reservoir ofHistoplasma capsulatum var.duboisii, the etiological agent of histoplasmosis duboisii (African histoplasmosis) is not yet known. We report the isolation ofH. capsulatum var.duboisii from soil admixed with bat guano and from the intestinal contents of a bat in a sandstone cave in a rural area, Ogbunike in Anambra State of Nigeria. Eight of 45 samples of soil admixed with bat guano yieldedH. capsulatum var.duboisii. Of the 35 bats belonging to the speciesNycteris hispida andTadirida pumila examined, only one (N. hispida) yielded this fungus from its intestinal contents. Identification of the isolates asHistoplasma was confirmed by exoantigen tests and by mating with tester strains ofH. capsulatum. In vitro conversion to large yeast from suggestive ofH. capsulatum var.duboisii was obtained on brain heart infusion agar supplemented with sheep blood and glutamine or cysteine. Pathogenicity tests with mice for all the isolates confirmed their identity by the demonstration of large yeast forms (8–15 µm in diameter) within giant cells in the infected tissues. Investigations on the possible occurrence of human infections in the area are in progress.A poster based on this work was presented at the 11th ISHAM Congress in Montreal, Canada (22–28 June 1991), La-Hoffman Roche, Basel, Switzerland kindly financed the trip of one of us (H.C.G) for the Congress.     

Genetic and chromosomal variation inPetunia hybrida plants regenerated from protoplast and callus cultures

Plants regenerated from callus cultures derived from leaf discs and mesophyll protoplasts ofPetunia hybrida cv. Rose of Heaven exhibit a high frequency of genetic and chromosomal variation. Of twelve leaf disc-derived plants examined, only three had the normal diploid chromosome number (2n=14) while seven were tetraploid and two were aneuploid (16 and 27 chromosomes). Of seventeen plants derived from two protoplasts, none had the diploid chromosome number. Most had 28 chromosomes, one 29, two 27, one 26 and one had variable numbers (14–28) in different root tip cells. In all cases aneuploidy was associated with developmental abnormality. In addition, heritable differences in growth, morphology and flower pigmentation were observed in callus-derived tetraploids and diploids, including one diploid which differed from parent plants in at least four characters. These results are discussed in terms of the importance ofPetunia in genetics research and for studies of somaclonal variation.     

Frankia-Alnus incana symbiosis: Effect of endophyte on nitrogen fixation and biomass production

The efficiency of different FinnishFrankia strains as symbionts onAlnus incana (L.) Moench was evaluated in inoculation experiments by measuring nitrogen fixation and biomass production. Since all available pure cultures ofFrankia are of the Sp⁻ type (sporangia not formed in nodules), but the dominant nodule endophyte ofA. incana in Finland is of the Sp⁺ type (sporangia formed in nodules), crushed nodules of thisFrankia type were included. The Sp⁻ pure cultures, whether originating fromA. incana orA. glutinosa, produced with one exception, similar biomass withA. incana. The highest biomass was produced with an American reference strain fromA. viridis crispa. Using Sp⁺ nodule homogenates fromA. incana as inoculum, the biomass production was only one third of that produced by Sp⁻ pure cultures from the same host. Hence, through selection of the endophyte it is possible to exert a considerable influence on the productivity ofAlnus incana.     

Production and harvesting of ionically wall-bound extensin from living cell suspension cultures

Cell-suspension cultures ofSpinacia andRosa accumulated a cell wall protein, extensin, in a form that was amenable to leaching from the surface of the living cells by a brief treatment with non-toxic salts. Cultures ofLycopersicon, Capsicum, Acer andFestuca did not accumulate this class of extensin. InSpinacia andRosa, optimum yields of leachable extensin were achieved from young cultures, in media at relatively low pH, by leaching with 0.1 M CaCl₂. Older cultures, pH values >6.5, and LaCl₃ or higher concentrations of CaCl₂ were less effective.Abbreviation TCA trichloroacetic acid     

Distribution of cells in different stages of the cell cycle and some age-related physiological characteristics in dependence on the dilution rate in chemostat cultures ofCandida utilis

Position of cells in their cell cycle was determined microscopically in chemostat cultures ofCandida utilis. Proportion of cells in phase G₁ decreased in a linear manner from 86% to 58% with dilution rate. Proportion of cells in phase S increased in the same range ofD from 5.6 to 13.5% and in the (G₂+M) phase from 8.4 to 28.5%, again linearly. Differential centrifugation was used to separate chemostat cultures to mother and daughter cells. Analyses showed that, relative to mother cells, daughter cells contain 2.1–11.9% more protein and 25.5–34.6% more RNA in dry matter. Their mass is 34.4–5.6% lower and volume is 154–19% smaller.     

Anatoxin-a concentration inAnabaena andAphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxicAnabaena-strains — a laboratory study

Anatoxin-a-concentration in cells ofAnabaena- andAphanizomenon-strains and in their growth media were studied in the laboratory in batch cultures at different temperatures, light fluxes, orthophosphate and nitrate concentrations and with different nitrogen sources for growth. Toxin concentrations were detected by HPLC. Also, the growth of the toxicAnabaena-strains was compared to that of a non-toxic one. The non-toxicAnabaena was never found to produce anatoxin-a. The amount of toxin in the cells of the toxic strains was high, often exceeding 1% of their dry weight. High temperature decreased the amount of the toxin regardless of growth. Growth limiting low and growth inhibiting high light decreased the amount of the toxin in the cells ofAnabaena-strains. The highest light flux studied did not limit the growth or decrease the level of the toxin in the cells ofAphanizomenon. Growth in N-free medium (i.e. N₂ fixation) showed that the cells contained more toxin than growth in N-rich medium. Orthophosphate concentration had no effect on toxin levels, although the lowest concentrations limited the growth of all strains studied. The toxic strains tolerated higher temperatures than the non-toxic one, but the non-toxic strain seemed to be more adjustable to high irradiance than the toxic ones. The yields (dry weight) of non-toxic and toxic strains differed significantly in different phosphate concentrations.     

An auto-inhibitor of zygote giant cell formation in Dictyostelium discoideum

The first observable event of sexual development in mated cultures (NC4x V12) of Dictyostelium discoideum is the appearance of zygote giant cells which are the product of cell fusions. The increase in the number of giant cells, which begins at about 21 h, is complete by 28 h. In this paper we show that a low molecular weight auto-inhibitor of zygote giant cell formation is present in 28-h cultures. The data presented suggest that this auto-inhibitor is a natural regulator of sexual development in D. discoideum.     

Amylostereum laevigatum associated with the Japanese horntail,Urocerus japonicus

The fungus associated with the Japanese horntail,Urocerus japonicus, in Kochi, Kagawa and Ehime Prefectures was studied. Cultures isolated from the mycangia of 113 adult females of the horntail showed the same cultural characteristics. Four of basidiocarps found on felled logs ofCryptomeria japonica were identifieds asAmylostereum laevigatum based on morphological characteristics. This was the first record ofA. laevigatum from Japan. The cultures isolated from the basidiocarps had the same cultural characteristics as those from the mycangia ofU. japonicus. One mycangial isolate produced basidiocarps on artificially inoculated stem segments ofCr. japonica after a 6-mo incubation and was identified asA. laevigatum. One isolate from the basidiocarps ofA. laevigatum and one from the mycangium ofU. japonicus were artificially inoculated into five trees each ofChamaecyparis obtusa andCr. japonica. The wood of all inoculated trees showed discoloration, with no difference in shape and pattern of discoloration between the two isolates. The inoculated fungi were reisolated from the areas of discoloration in the inoculated trees.     

Dihexyl sulfosuccinate biodegradation by mixed cultures

The primary biodegradation of dihexyl sulfosuccinate has been demonstrated using two single cultures and three mixed bacterial cultures ofComamonas terrigena. Primary biodegradation was found in all variants. The highest biotransformation rate and efficiency was observed in one of the single strains. No synergistic effect on degradation was found in mixed cultures.