Kinetic study of the toxicity of zinc and lead ions to the heavy metals accumulating fungus Paecilomyces marquandii

Studies have been carried out to determine the toxicity of zinc and lead ions to germinating spores and hyphal growth of heavy metal accumulating fungus Paecilomyces marquandii (former Verticillium marquandii). Inhibitive concentration (IC₅₀) of zinc and lead ions was assayed by three different methods: image analysis, nephelometric on-line measurement and microcalorimetry. A kinetic model of spore germination and germ tube elongation was formulated and used as an auxiliary tool to determine IC₅₀ values upon image analysis data. The inhibitive effect of Zn²⁺ and Pb²⁺ to P. marquandii spores was mathematically described by the Edwards equation. Comparing the obtained IC₅₀ values, lead ions occurred to be more toxic to the germinating spores of P. marquandii than zinc ions (2.80 and 5.20 mM, respectively), although zinc ions induced a more significant delay in the development of the hyphae (13.84 h for 5 mM of Zn²⁺ and 9.30 h for 5 mM of Pb²⁺), which was demonstrated by the lengthened lag-phase (spore-swelling phase).     

Responses to accelerated heavy ions of spores ofBacillus subtilis of different repair capacity

Summary Inactivation, mutagenesis of histidine reversion and the involvement of DNA repair were studied in spores ofBacillus subtilis irradiated with heavy ions at LBL, Berkeley and GSI, Darmstadt. Five groups of ions (from boron to uranium) were used with residual energies from 0.2 MeV/u up to 18.6 MeV/u; in addition, carbon ions were used with a residual energy of 120 MeV/u. Action cross sections of both inactivation and mutagenesis show a similar dependence on ion mass and energy: for lighter ions (Z 10), the lethal response is nearly energy independent (Z = 10) or decreasing with energy (Z 6); these light ions, up to 18.6 MeV/u, induce hardly any mutations. For heavier ions (Z 26), the lethal as well as the mutagenic responses increase with ion mass and energy up to a maximum or saturation. The efficiency of DNA repair to improve survival and the mutagenic efficiency per lethal event, both, increase with ion energy up to a saturation value which, depending on strain and endpoint, either roughly coincides with the X-ray value or is smaller than that after X-ray treatment. For repair based on recombination events, the increase in the survival effects with ion energy is more pronounced than for that based on repair replication. At energies of 1 MeV/u or below, neither DNA repair nor mutation induction appear to be significant. The results support previous suggestions on the importance of the radial distribution of the energy around the ion track in biological action cross section and the evidence that the entire core of the spore represents the sensitive site in responses to heavy ions.     

Phytochelatins and heavy metal tolerance

The induction and heavy metal binding properties of phytochelatins in heavy metal tolerant (Silene vulgaris) and sensitive (tomato) cell cultures, in water cultures of these plants and in Silene vulgaris grown on a medieval copper mining dump were investigated. Application of heavy metals to cell suspension cultures and whole plants of Silene vulgaris and tomato induces the formation of heavy metal–phytochelatin-complexes with Cu and Cd and the binding of Zn and Pb to lower molecular weight substances. The binding of heavy metal ions to phytochelatins seems to play only a transient role in the heavy metal detoxification, because the Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Free heavy metal ions were not detectable in the extracts of all investigated plants and cell cultures. Silene vulgaris plants grown under natural conditions on a mining dump synthesize low molecular weight heavy metal binding compounds only and show no complexation of heavy metal ions to phytochelatins. The induction of phytochelatins is a general answer of higher plants to heavy metal exposition, but only some of the heavy metal ions are able to form stable complexes with phytochelatins. The investigation of tolerant plants from the copper mining dump shows that phytochelatins are not responsible for the development of the heavy metal tolerant phenotypes.     

Cellular and subcellular effects of very heavy ions

The biological effects of irradiation with ions of masses larger than 40 and energies up to 20 MeV per atomic mass unit are reviewed. The objects are viruses, bacterial spores, yeast and mammalian cells. Experimental parameters include loss of colony forming ability, induction of mutants, chromosomal aberrations, cell cycle progression, inhibition of biochemical activities and the formation of strand breaks. Some of the pertinent physical questions--e.g. track structure--are also discussed. It is shown that with very heavy ions the biological effectiveness is no longer unambiguously related to a single parameter like l.e.t. or Z*2/beta 2 but depends strongly on ion energy. This points to the importance of far-reaching delta-electrons. The analysis indicates also that even with very high l.e.t., cells are not killed by the passage of a single particle through their nucleus. Possible implications of the findings for fundamental radiation biology are outlined.     

Artificial wind-gust liberation of microbial bioaerosols previously deposited on plants

Summary A bioaerosol research chamber was constructed and used to evaluate wind-gust release of previously depositedPseudomonas syringae, spores ofBacillus subtilis var.niger, and fluorescent microspheres (FM) on oat plants, and the airborne survival of the releasedP. syringae. Observations of wind gusts on the releasedBacillus spores and FM showed they had similar particle size distributions and therefore FM could act as bacterial sized surrogates in particle dispersion. Microscopic examination of the released FM containing particles revealed that 84% were associated with either fungal spores and hyphae were epiphytic on the plant, or with plant and soil debris. The release rate ofBacillus spores decreased as the number of gusts experienced by the plants increased, with a greater proportion of larger particles removed. The larger the particle associated with the releasedP. syringae, the longer the bacteria survived.     

Temperature Dependency of the Mutagenic Action of N-Methyl-N′-nitro-N-nitrosoguanidine in Streptomyces cacaoi

The effectiveness of N-methyl-N′-nitro-N-nitrosoguanidine (NG) in inducing mutations in Streptomyces cacaoi was demonstrated with two systems: reversion to prototrophy of an isoleucine-requiring mutant and the induction of forward mutation of a prototrophic strain. An optimal condition for yielding a high frequency mutation was set as follows: Treatment of spores with 1 mg/ml of NG in 0.016 m phosphate buffer, pH 6, at 42°C for 60 min. In Streptmyces cacaoi, the mutagenesis depended highly on the temperature of NG treatment; elevation of the treatment temperature resulted higher frequency of mutation. Only a little dependency on temperature was shown in Escherichia coli, but the dependency was also observed when the Streptomyces was treated with ethyl methanesulfonate. The significance of these results for considering mechanism of NG action was discussed.     

低能重离子在作物种胚内射程分布的研究技术

我国科学工作者率先将低能重离子束成功地应用于作物诱变育种,并建立了能量,质量和电荷三因子作用机制体系,但至今有关理论射程很短的低能重离子注入生物体后如何通过信息传递而诱发生物学效应的机理尚不完全清楚,低能重离子在作物种胚内的实际射程分布迄今仍是一个颇有争议的热点问题,而该项研究就直接触及低能离子束与生物组织细胞的原初作用机制,应用低能放射性束或具有可探测放射性的核反应产物,通过超薄切片和逐层分析测定,即可定量计算不同能量的低能离子束在作物种胚内的射程分布,本文还探讨了激光共聚焦扫描显微镜,原子力显微镜,X-射线能谱分析技术,单粒子微束技术和图像处理等技术途径在该项研究中的应用。     

Effects of heavy ion to the primary [correction of rimary] culture of mouse brain cells.

To investigate effects of low dose heavy particle radiation to CNS system, we adopted mouse neonatal brain cells in culture being exposed to heavy ions by HIMAC at NIRS and NSRL at BNL. The applied dose varied from 0.05 Gy up to 2.0 Gy. The subsequent biological effects were evaluated by an induction of apoptosis and neuron survival focusing on the dependencies of the animal strains, SCID, B6, B6C3F1, C3H, used for brain cell culture, SCID was the most sensitive and C3H the least sensitive to particle radiation as evaluated by 10% apoptotic criterion. The LET dependency was compared with using SCID and B6 cells exposing to different ions (H, C, Ne, Si, Ar, and Fe). Although no detectable LET dependency was observed in the high LET (55-200 keV/micrometers) and low dose (<0.5 Gy) regions. The survivability profiles of the neurons were different in the mouse strains and ions. In this report, a result of memory and learning function to adult mice after whole-body and brain local irradiation at carbon ion and iron ion.     

Heavy ion effects on yeast cells: induction of canavanine-resistant mutants

The induction of forward mutations (resistance to canavanine) by heavy ion bombardment was investigated in wild type haploid yeast Saccharomyces cerevisiae. Accelerated ions of argon, titanium, nickel, krypton, xenon, lead and uranium with specific energies between 1.7 and 9.25 MeV/u were obtained from the UNILAC machine at the Gesellschaft für Schwerionenforschung, Darmstadt/Germany. LET-values ranged from 1200 to about 15 000 keV/microns. There was no unequivocal dependence of mutation induction cross section on either LET or Z*2/beta 2, but also a prominent influence of ion specific energy. This is explained by the action of long-ranging delta-electrons.     

INFLUENCE OF IONIC CONDITIONS ON CELL DIFFERENTIATION AND MORPHOGENESIS OF THE CELLULAR SLIME MOLDS

Effects of various ionic conditions on the development of the cellular slime molds D. discoideum and D. mucoroides were studied. A certain concentration of lithium ions (7 mM) promoted differentiation of the stalk cells and conversely inhibited formation of the spores. The presence of calcium and magnesium ions was needed for Li to manifest its specific effect. A high concentration of Ca (100-120 mM) also facilitated differentiation of the stalk cells. On the other hand, fluoride ions stimulated considerable formation of spores at 15 mM. In the absence of divalent cations, sodium ions inhibited morphogenesis and cell differentiation proportionately with its concentration, and complete inhibition was obtained at 20 mM. The inhibitory effect of Na was nullified by addition of small amounts of Ca. Possible mechanisms by which these ions exert their influences on development of this organism were discussed.     

Induction of HPRT- mutants in Chinese hamster V79 cells after heavy ion exposure

The induction of resistance to 6-thioguanine by heavy ion exposure was investigated with various accelerated ions (oxygen-uranium) up to linear energy transfer (LET) values of about 15000 keV/µm.31 y Survival curves are exponential with fluence; mutation induction shows a linear dependence. Cross-sections (_i: inactivation, _m: mutation) were derived from the respective slopes. Generally, _i rises over the whole LET range, but separateas into different declining curves for single ions with LET values above 200 keV/µm. Similar behaviour is seen for _m. The new SIS facility at GSI, Darmstadt, makes it possible to study the effects of ions with the same LET but very different energies and track structures. Experiments using nickel and oxygen ions (up to 400 MeV/u) showed that inactivation cross-sections do not depend very much on track structure, i.e. similar values are found with different ions at the same LET. This is not the case for mutation induction, where very energetic ions display considerably smaller induction cross-sections compared with low-energy ions of identical LET. Preliminary analyses using the polymerase chain reaction (PCR) demonstrate that even heavy ions cause small alterations (small deletions or base changes). The proportion of the total deletions seems to increase with LET.Submitted paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

Hydrogen gas accelerates thermal inactivation ofClostridium botulinum 113B spores

Summary Heat inactivation ofClostridium botulinum spores was accelerated in atmospheres containing hydrogen gas. Hydrogen gas also moderately increased the thermal destruction ofBacillus spores. Hydrogen gas may react with components inC. botulinum spores such as transition metals producing hydrogen ions or hydrides, which destroy essential spore components. Thermal processing in modified atmospheres may have applications in food processing and in sterilization of medical supplies.     

Heavy metal-mediated activation of the rat Cu/Zn superoxide dismutase gene via a metal-responsive element

The Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced in the course of biological oxidations. When placed under the control of the rat SOD1 gene promoter and transfected into human HepG2 hepatoma cells, the activity of a chloramphenicol acetyltransferase reporter gene was found to increase three- to four-fold in the presence of heavy metals (cadmium, zinc and copper). Functional analysis of mutant derivatives of the SOD1 gene promoter and the use of a heterologous promoter system confirmed that the induction of the SOD1 gene by metal ions requires a metal-responsive element (MRE) located between positions −273 and −267 (GCGCGCA). It was also shown by gel mobility shift assays that an MRE binding protein is induced by the exposure of the human liver cell line HepG2 to heavy metals. These results suggest that the MRE participates in the induction of the SOD1 gene by heavy metals. Received: 5 February 1999 / Accepted: 21 May 1999     

Reduction of Methionine Sulfoxide by Plants

A relaxed (rel) mutant was found among thirty spontaneous thiopeptin-resistant isolates of Streptomyces antibioticus strain 3720, an actinomycin-producing strain, which showed severely reduced ability to accumulate ppGpp during a nutritional shift-down. The pool size of GTP decreased markedly in the parental strain, but to a lesser extent in the rel mutant. The rel mutant did not show the induction of an enzyme, phenoxazinone synthase, which is involved in the biosynthesis of actinomycin. No negative effect of the rel mutation was observed on a constitutive enzyme, kynurenine formamidase, which also plays a role in actinomycin synthesis. The mutant also failed to produce melanin, but still retained the ability to form aerial mycelium and spores, although the onset of the formation of aerial mycelium was markedly delayed. Neither the phenoxazinone synthase activity nor the kynurenine formamidase activity was affected by ppGpp in vitro. It is suggested tha the stringent response (ppGpp) may be generally essential for the induction of enzymes involved in secondary metabolism.     

Assessment of arbuscular mycorrhizal fungi diversity in the rhizosphere of <Emphasis Type="Italic">Viola calaminaria</Emphasis> and effect of these fungi on heavy metal uptake by clover

 The ability of arbuscular mycorrhizal (AM) fungi from a metal-tolerant plant (Viola calaminaria, violet) to colonise and reduce metal uptake by a non-tolerant plant (Trifolium subterraneum, subterranean clover) in comparison to a metal-tolerant AM fungus isolated from a non-tolerant plant was studied. AM spores from the violet rhizosphere and from violet roots were characterised by polymerase chain reaction (PCR) amplification of the SSU rDNA, and sequencing. Subterranean clover was grown in pots containing a soil supplemented with Cd and Zn salts and inoculated either with a mixture of spores extracted from the violet rhizosphere or with spores of a Cd-tolerant Glomus mosseae P2 (BEG 69), or non-inoculated. The diversity of fungi, including AM fungi, colonising clover roots was assessed and analysed using terminal-restriction fragment length polymorphism. At least four different Glomus species were found in the violet rhizosphere. After 8 weeks in a growth chamber, colonisation of clover roots with spores from the violet rhizosphere increased Cd and Zn concentrations in clover roots without significantly affecting the concentrations of metals in the shoot and plant growth. G. mosseae P2 reduced plant growth and slightly increased the Cd concentration. Only one AM fungus (Glomus b) from the violet rhizosphere colonised clover roots, but other fungi were present. AM fungi from heavy metal-contaminated soils and associated with metal-tolerant plants may be effective in accumulating heavy metals in roots in a non-toxic form. Accepted: 7 July 2000     

The Beltsville method for soilless production of vesicular-arbuscular mycorrhizal fungi

A low-cost, low-maintenance system for soilless production of vesicular-arbuscular mycorrhizal (VAM) fungus spores and inoculum was developed and adapted for production of acidophilic and basophilic isolates. Corn (Zea mays) plants were grown with Glomus etunicatum, G. mosseae or Gigaspora margarita in sand automatically irrigated with modified Hoagland's solution. Sand particle size, irrigation frequency, P concentration, and buffer constituents were adjusted to maximize spore production. Modified half-strength Hoagland's solution buffered with 4-morpholine ethane-sulfonic acid (MES) automatically applied 5 times/day resulted in production of 235 G. etunicatum spores/g dry wt. of medium (341000 spores/pot) and 44 G. margarita spores/g dry wt. of medium (64800 spores/pot). For six basophilic isolates of G. mosseae, CaCO₃ was incorporated into the sand and pots were supplied with the same nutrient solution as for acidophilic isolates. The increased pH from 6.1±0.2 to 7.2±0.2 resulted in spore production ranging from 70 to 145 spores/g dry wt. (102000–210000 spores/pot). Spore production by all isolates grown in the soilless sand system at Beltsville has exceeded that of traditional soil mixtures by 32–362% in 8–12 weeks.     

A Possible Role for Ferrous Complexes in Fungal Disease Suppression: Glume Blotch and Net Blotch of Cereals

Germination of spores of Septoria nodorum and Pyrenophora teres was inhibited and germ-tube growth in germinated spores was reduced b y ferrous ions complexed with a number of cheiating agents. No such inhibition was observed with ferric complexes and none of the chelating agents in the desferri form was toxic to the fungi. The germination mechanism in spores suspended in ferrous-2,3-dihydroxybenzoic acid (Fe¹¹-DHBA) for 24 h could not subsequently be released to any great extent by incubation with ethylenediamjne-di (o-hydroxyphenyl-acetic acid) (EDDHA). Lesion development by the fungi in the presence of ferrous complexes on detached leaves of host plants was almost totally suppressed, but compounds which preferentially chelate ferric ions, used in the desferri form significantly stimulated lesion development by S. nodorum on wheat leaves. Cermination, appressorium formation and lesion development on leaves of host plants were also significantly reduced by Fe¹¹-DHBA when plants were sprayed to run-off up to 5 days prior to inoculation. Disease development and subsequent 1000 grain weight loss were reduced by approximately 50 percent in wheat and barley plants when the flag leaves were treated with Fe¹¹-DHBA (5 × 10^?4 M) prior to inoculation with S. nodorum and P. teres respectively, compared with inoculated, untreated plants.     

Heavy ion-induced DNA double-strand breaks with yeast as a model system

Cells of diploid yeast,Saccharomyces cerevisiae, were exposed to a variety of energetic heavy ions (provided by the UNILAC facility at the Gesellschaft für Schwerionenforschung, GSI),²⁴¹Am-particles and 80-keV x-rays after which they were assessed for DNA double-strand breaks (DSB) using either the neutral sedimentation or the pulsed-field gel electrophoresis (PFGE) technique. Both yielded comparable results. The DSB production cross-sections are compared with inactivation studies performed for the same cells under identical conditions. The measurements show that with lighter ions DSB induction cross-sections increase with linear energy transfer (LET), but the situation is less clear with the heavier ions. A close parallelism was found between DSB induction and cell inactivation in these yeast cells.Submitted paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

Mutation induction in Neurospora crassa incubated in mice and rats

Summary When Neurospora crassa conidia were injected into the peritoneal cavity of untreated mice or rats and kept there for more than 24 hours, the ad-3 mutation frequency among the surviving conidia increased sharply, more so in rats than in mice. This increase in the ad-3 mutation frequency was attributed both to direct cellular contact between conidia and mammalian cells and to macromolecules already present in untreated animals. Conidia enclosed in dialysis tubing or in diffusion chambers placed surgically in the peritoneal cavity had a much lower frequency of ad-3 mutations than conidia injected into the peritoneal cavity as a suspension. This was interpreted as indicating that the major fraction of mutations are mediated through a cellular contact.To determine whether the dialysis bags were permeable to mutagens, a comparison was made between the mutation frequencies obtained with conidia placed in dialysis bags and with conidia distributed at random throughout the peritoneal cavity in host animals treated with methyl methanesulfonate (MMS). MMS (100 mg/kg) was injected into the tail vein 8 hours after the conidia were placed in the animals. Ten hours after the injection of the mutagen, the conidia were recovered and analyzed for the induction of ad-3 mutations. The MMS-induced mutation frequency was the same among both groups of conidia, demonstrating that the dialysis bags did not become impermeable to small molecules during the time of incubation in the animal.In the host-mediated assay an indiactor organism is injected into the peritoneal cavity of an animal which is then treated with a chemical or its metabolites, to assay for mutagenicity. The present experiments show that the increased mutation frequency induced in the indicator organism after intraperitoneal injection and incubation may give false positive results in the host-mediated assay unless a comparison is made with suitable untreated controls.Autopsies of animals 24 days after intraperitoneal inoculation with Neurospora conidia, and sectioning and staining of various organs (Malling and Cosgrove, 1970) showed that some conidia were still localized in various organs, even though essentially all of them had been inactivated 96 hours after injection. The inactivation of these fungal spores may result from an enzymatic degradation of their DNA, mediated by the host, and halting this process prematurely may result in the induction of recessive-lethal mutations. Thus these studies also suggest that one of the important defense mechanisms of higher animals against infectious organisms may be the induction of mutations.Research sponsored by the National Cancer Institute, National Institutes of Health, and by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation.     

The specific transport system for lysine is fully inhibited by ammonium in Penicillium chrysogenum: An ammonium-insensitive system allows uptake in carbon-starved cells

The regulation exerted by ammonium and other nitrogen sources on amino acid utilization was studied in swollen spores of Penicillium chrysogenum. Ammonium prevented the L-lysine, L-arginine and L-ornithine utilization by P. chrysogenum swollen spores seeded in complete media, but not in carbon-deficient media. Transport of L-[¹⁴C]lysine into spores incubated in presence of carbon and nitrogen sources was fully inhibited by ammonium ions (35 mM). However, in carbon-derepressed conditions (growth in absence of sugars, with amino acids as the sole carbon source) L-[¹⁴C]lysine transport was only partially inhibited. Competition experiments showed that L-lysine (1 mM) inhibits the utilization of L-arginine, and vice versa, L-arginine inhibits the L-lysine uptake. High concentrations of L-ornithine (100 mM) prevented the L-lysine and L-arginine utilization in P. chrysogenum swollen spores. In summary, ammonium seems to prevent the utilization of basic amino acids in P. chrysogenum spores by inhibiting the transport of these amino acids through their specific transport system(s), but not through the general amino acid transport system that is operative under carbon-derepression conditions.