PIXE-electrophoresis shows starving collembolan reallocates protein-bound metals.

One of multiple functions of metalloproteins is to provide detoxification to excess metal levels in organisms. Here we address the induction and persistence of a range of low to high molecular weight copper- and zinc binding proteins in the collembolan species Tetrodontophora bielanensis exposed to copper- and zinc-enriched food, followed by a period of recovery from metal exposure, in absence and presence of food. After 10 days of feeding copper and zinc contaminated yeast, specimens were either moved to ample of leaf litter material from their woodland stand of origin or starved (no food offered). The molecular weight distribution of metal binding proteins was determined by native polyacryl gel electrophoresis. One gel was stained with Comassie brilliant blue and a duplicate gel dried and scanned for the amount of copper and zinc by particle-induced X-ray emission. Specimens exposed to copper and recovered from it with ample of food had copper bound to two groups of rather low molecular weight proteins (40-50 kDa) and two of intermediate size (70-80 kDa). Most zinc in specimens from the woodland stand was bound to two large proteins of about 104 and 106 kDa. The same proteins were holding some zinc in metal-exposed specimens, but most zinc was found in proteins <40 kDa in size. Specimens recovered from metal exposure in presence of ample of food had the same distribution pattern of zinc binding proteins, whereas starved specimens had zinc as well as copper mainly bound to two proteins of 8 and 10 kDa in size. Thus, the induction and distribution of copper- and zinc-binding proteins depend on exposure conditions, and the presence of low molecular weight binding proteins, characteristic of metallothioneins, was mainly limited to starving conditions.

Elemental composition of arbuscular mycorrhizal fungi at high salinity.

We investigated the elemental composition of spores and hyphae of arbuscular mycorrhizal fungi (AMF) collected from two saline sites at the desert border in Tunisia, and of Glomus intraradices grown in vitro with or without addition of NaCl to the medium, by proton-induced X-ray emission. We compared the elemental composition of the field AMF to those of the soil and the associated plants. The spores and hyphae from the saline soils showed strongly elevated levels of Ca, Cl, Mg, Fe, Si, and K compared to their growth environment. In contrast, the spores of both the field-derived AMF and the in vitro grown G. intraradices contained lower or not elevated Na levels compared to their growth environment. This resulted in higher K:Na and Ca:Na ratios in spores than in soil, but lower than in the associated plants for the field AMF. The K:Na and Ca:Na ratios of G. intraradices grown in monoxenic cultures were also in the same range as those of the field AMF and did not change even when those ratios in the growth medium were lowered several orders of magnitude by adding NaCl. These results indicate that AMF can selectively take up elements such as K and Ca, which act as osmotic equivalents while they avoid uptake of toxic Na. This could make them important in the alleviation of salinity stress in their plant hosts.

Phosphorus availability influences elemental uptake in the mycorrhizal fungus Glomus intraradices, as revealed by particle-induced X-ray emission analysis.

We investigated element accumulation in the arbuscular mycorrhizal fungus Glomus intraradices. Fungal spores and mycelia growing in monoxenic cultures were analyzed. The elemental composition was quantified using particle-induced X-ray emission (PIXE) in combination with scanning transmission ion microscopy. In the spores, Ca and Fe were associated mainly with the spore wall, while P and K showed patchy distributions and their concentrations were correlated. Excess of P in the hyphal growth medium increased the P and Si concentrations in spores and increased the K/Ca ratio in spores. Increased P availability decreased the concentration of Zn and Mn in spores. We concluded that the availability of P influences the uptake and accumulation of several elements in spores. It is demonstrated that PIXE analysis is a powerful tool for quantitative analysis of elemental accumulation in fungal mycelia.

Quantification of lithium at ppm level in geological samples using nuclear reaction analysis.

Proton-induced reaction (p,α) is one type of nuclear reaction analysis (NRA) suitable especially for light element quantification. In the case of lithium quantification presented in this work, accelerated protons with an energy about of 850 keV were used to induce the 7Li(p,α)4He reaction in standard reference and geological samples such as tourmaline and other Li-minerals. It is shown that this technique for lithium quantification allowed for measurement of concentrations down below one ppm. The possibility to relate the lithium content with the boron content in a single analysis was also demonstrated using tourmaline samples, both in absolute concentration and in lateral distribution. In addition, Particle induced X-ray emission (PIXE) was utilized as a complementary IBA technique for simultaneous mapping of elements heavier than sodium.

Nuclear microscopy: a tool for imaging elemental distribution and percutaneous absorption in vivo.

Nuclear microscopy is a technique based on a focused beam of accelerated particles that has the ability of imaging the morphology of the tissue in vivo and of producing the correspondent elemental maps, whether in major, minor, or trace concentrations. These characteristics constitute a strong advantage in studying the morphology of human skin, its elemental distributions and the permeation mechanisms of chemical compounds. In this study, nuclear microscopy techniques such as scanning transmission ion microscopy and particle induced X-ray emission were applied simultaneously, to cryopreserved human skin samples with the purpose of obtaining high-resolution images of cells and tissue morphology. In addition, quantitative elemental profiling and mapping of phosphorus, calcium, chlorine, and potassium in skin cross-sections were obtained. This procedure accurately distinguishes the epidermal strata and dermis by overlapping in real time the elemental information with density images obtained from the transmitted beam. A validation procedure for elemental distributions in human skin based on differential density of epidermal strata and dermis was established. As demonstrated, this procedure can be used in future studies as a tool for the in vivo examination of trans-epidermal and -dermal delivery of products.

A nuclear geochemical analysis system for boron quantification using a focused ion beam.

Ion beam analysis has for decades been used as a tool for geochemical analysis of trace elements using both X-rays (particle induced X-ray emission) and nuclear reaction analysis. With the geoanalytical setup at the Lund Ion Beam Analysis Facility, the boron content in geological samples with a spatial resolution of 1 µm is determined through nuclear reaction analysis. In the newly upgraded setup, a single detector has been replaced by a double sided silicon strip detector with 2048 segments. After optimization, boron content in geological samples as low as 1 µg g-1 can be measured.

Temporal changes in the elemental composition of Rhizopogon rhizomorphs during colonization of patches with fresh organic matter or acid-washed sand.

A laboratory experiment was performed to estimate the elemental composition of rhizomorphs of an ectomycorrhizal (EM) fungus growing in a patchy environment. Successive samples of Rhizopogon rhizomorphs, located adjacent to patches with organic matter or patches with acid-washed sand, were taken over a period of 45 d. The mass per unit area was analyzed with scanning transmission ion microscopy (STIM), and the elemental content of elements heavier than Mg were analyzed with particle induced X-ray emission (PIXE). Rhizomorphs associated with the organic matter on average were three times heavier per unit area than rhizomorphs associated with sand. The Ca concentration (mg g(-1)) increased in rhizomorphs adjacent to patches with sand, while it decreased in rhizomorphs adjacent to patches with organic matter. Fe concentration was higher in rhizomorphs adjacent to patches with sand. We concluded that the EM fungus responded to the organic matter by producing larger rhizomorphs, rather than increasing the concentration of elements in the rhizomorphs, to improve the transport of elements to the roots. The elemental composition of rhizomorphs varied considerably over time, and the accumulation of Ca in rhizomorphs in the sand-filled compartments could be the effect of acropetal flow of solutes from the plant roots toward the mycelial front.

A1M/α1-microglobulin is proteolytically activated by myeloperoxidase, binds its heme group and inhibits low density lipoprotein oxidation.

α1-microglobulin (A1M) is a 26 kDa plasma and tissue protein with reductase activity and radical- and heme-binding anti-oxidative functions. In addition, exposure of A1M to hemoglobin has been shown to induce proteolytic elimination of a C-terminal tetrapeptide yielding a heme-degrading form, truncated A1M (t-A1M). Myeloperoxidase (MPO), a heme-containing enzyme that catalyzes the production of free radicals and hypochlorite, is released by neutrophils during the inflammatory response to bacterial infections. MPO-induced low density lipoprotein (LDL)-oxidation in blood has been suggested as a causative factor in atherosclerosis. In this study we have hypothesized that A1M interacts with MPO in a similar mode as with hemoglobin, and is a regulator of its activity. The results show that A1M is proteolytically cleaved, with formation of t-A1M, after exposure to MPO, and that t-A1M contains iron and heme-degradation products. The reaction is dependent of pH, time and concentration of substrates and a pH-value around 7 is shown to be optimal for cleavage. Furthermore, A1M inhibits MPO- and hydrogen peroxide-induced oxidation of LDL. The results suggest that A1M may have a role as an inhibitor of the damaging effects of the neutrophil respiratory burst on bystander tissue components.

Tit for tat? A mycorrhizal fungus accumulates phosphorus under low plant carbon availability.

The exchange of carbohydrates and mineral nutrients in the arbuscular mycorrhizal (AM) symbiosis must be controlled by both partners in order to sustain an evolutionarily stable mutualism. Plants downregulate their carbon (C) flow to the fungus when nutrient levels are sufficient, while the mechanism controlling fungal nutrient transfer is unknown. Here, we show that the fungus accumulates nutrients when connected to a host that is of less benefit to the fungus, indicating a potential of the fungus to control the transfer of nutrients. We used a monoxenic in vitro model of root organ cultures associated with Glomus intraradices, in which we manipulated the C availability to the plant. We found that G. intraradices accumulated up to seven times more nutrients in its spores, and up to nine times more in its hyphae, when the C pool available to the associated roots was halved. The strongest effect was found for phosphorus (P), considered to be the most important nutrient in the AM symbiosis. Other elements such as potassium and chorine were also accumulated, but to a lesser extent, while no accumulation of iron or manganese was found. Our results suggest a functional linkage between C and P exchange.

Elemental composition in vesicles of an arbuscular mycorrhizal fungus, as revealed by PIXE analysis.

We investigated element accumulation in vesicles of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, extracted from the roots of inoculated leek plants. The elemental composition (elements heavier than Mg) was quantified using particle-induced X-ray emission (PIXE), in combination with scanning transmission ion microscopy (STIM). In vesicles, P was the most abundant of the elements analysed, followed by Ca, S, Si and K. We analysed 12 vesicles from two root systems and found that the variation between vesicles was particularly high for P and Si. The P content related positively to Si, Zn and K, while its relation to Cl fitted to a negative power function. Vesicle transects showed that P and K were present in central parts, while Ca was present mainly near the vesicle surfaces. The results showed that P is an important part (0.5% of the dry weight) of the vesicle content and that the distribution of some elements, within mycelia, may be strongly correlated.

Tolerance to proton irradiation in the eutardigrade Richtersius coronifer--a nuclear microprobe study.

PURPOSE: The tardigrade Richtersius coronifer has previously been shown to tolerate very high doses of low linear energy transfer (low-LET) radiation (gamma rays). The purpose of this study was to extend our knowledge on radiation tolerance in this species by investigating the dose-response to high-LET radiation in terms of protons. MATERIALS AND METHODS: Dehydrated tardigrades of the species R. coronifer were irradiated with 2.55 MeV (megaelectronvolts) protons at doses ranging from 500 gray (Gy) to 15,000 Gy, to investigate the dose-viability relationship. In addition, a focused proton microbeam was utilised to determine the areal mass distribution, using the ion beam analytical technique STIM (Scanning Transmission Ion Microscopy). RESULTS: The experiment suggests that R. coronifer is unaffected by doses of proton irradiation up to 10,000 Gy, but shows very little viability at higher doses. The STIM analysis revealed that the thickness of the dehydrated tardigrades exceeds 150 microm, and that a fraction of the protons may not be fully absorbed. CONCLUSION: Our results are in line with previous studies of exposure to high-LET radiation in tardigrades, indicating that these animals are equally or even more tolerant to high-LET compared to low-LET gamma radiation. The physiological background to this remarkable result is currently unknown, but deserves investigation.

Bystander cell death and stress response is inhibited by the radical scavenger α(1)-microglobulin in irradiated cell cultures.

Alpha-particle irradiation of cells damages not only the irradiated cells but also nontargeted bystander cells. It has been proposed that the bystander effect is caused by oxidants and free radicals generated by the radiation. Recent studies have shown that α(1)-microglobulin protects against cell damage caused by oxidants and free radicals. Using a novel experimental system that allows irradiation of 0.02% of a human hepatoma monolayer, leaving 99.98% as bystander cells, we investigated the influence of oxidative stress and the cell-protective effects of α(1)-microglobulin during α-particle irradiation. The results showed an increase in cell death in both irradiated cells and bystander cells. A significant increase in apoptosis, oxidation markers and expression of the stress response genes heme oxygenase 1, superoxide dismutase, catalase, glutathione peroxidase 1, p21 and p53 were observed. Addition of α(1)-microglobulin reduced the amount of dead cells and inhibited apoptosis, formation of oxidation markers, and up-regulation of stress response genes. The results emphasize the role of oxidative stress in promoting bystander effects. Furthermore, the results suggest that α(1)-microglobulin protects nonirradiated cells by eliminating oxidants and free radicals generated by radiation and imply that α(1)-microglobulin can be used in radiation therapy of tumors to minimize damage to surrounding tissues.

The elemental content in the mycelium of the ectomycorrhizal fungus Piloderma sp. during the colonization of hardened wood ash.

Piloderma sp., a wood ash-colonizing ectomycorrhizal (EM) fungus, was grown symbiotically with Norway spruce in microcosms which contained granules of hardened wood ash. Mycelium close to the granules was sampled 3 times over a period of 11 weeks and the elemental content was investigated with particle induced X-ray emission. Mycelium from microcosms without wood ash was used as controls. The contents of P and K were similar in mycelium growing close to wood ash granules to those in control mycelium, while the Ca content increased from 23+/-21 mg g(-1) in controls to 63+/-8 mg g(-1) in mycelium growing close to wood ash granules. The Ca content was also increased in other parts of the mycelium more distant from the wood ash. Piloderma sp. may have a role in the short-term storage of Ca released from wood ash, rather than in releasing and storing P.

PIXE analysis to estimate the elemental composition of ectomycorrhizal rhizomorphs grown in contact with different minerals in forest soil.

The aim of this study was to investigate possible interactions between minerals and ectomycorrhizal (EM) mycelia. Fungal rhizomorphs growing in association with apatite and/or biotite were sampled both from a laboratory experimental system (Rhizopogon sp. and Pinus muricata) and from mesh bags buried in forest soil in the field. The elemental composition of the samples was analyzed with particle-induced X-ray emission (PIXE). Many EM rhizomorphs associated with apatite in laboratory systems and in mesh bags contained larger amounts of Ca (mean ranges between 12 and 31 mg Ca g(-1)) than similar rhizomorphs connected to acid-washed sand (range 0.3-3.5 mg Ca g(-1)). Ca originating from apatite was deposited as calcium oxalate crystals on the surface of the rhizomorphs. EM mycelium produced in mesh bags had a capacity to mobilize 0.6 mg P kg(-1) year(-1) from apatite-amended sand (which is 0.04% of the added apatite). A high concentration of K in some rhizomorphs (up to 11 mg K g(-1)) suggests that these fungi are good accumulators of K and may have a significant role in transporting K to trees.

Elemental composition of ectomycorrhizal mycelia identified by PCR-RFLP analysis and grown in contact with apatite or wood ash in forest soil.

Abstract The aim of this study was to identify ectomycorrhizal species with a potential to release elements from apatite and wood ash and accumulate them in the mycelia. Fungal rhizomorphs and mycelia were sampled from sand-filled mesh bags with or without amendment of apatite or wood ash. The mesh bags were buried in forest soil in the field for 13 or 24 months. Elemental composition of the samples was analyzed with particle-induced X-ray emission and the fungus was identified by polymerase chain reaction-restriction fragment length polymorphism analysis of the ITS-region of ribosomal DNA. The majority of rhizomorphs and mycelia collected from the mesh bags were of mycorrhizal origin with Paxillus involutus being the most common species (31%). Other identified species were Thelephora terrestris, Suillus granulatus and Tylospora fibillosa. S. granulatus contained 3-15 times more K (3 mg g(-1)) than the other species and had large calcium-rich crystals deposited on the surface of rhizomorphs when grown in contact with apatite. P. involutus contained the largest amount of Ca (2-7 mg g(-1)). Wood ash addition increased the amount of Ti, Mn and Pb in the rhizomorphs while apatite addition increased the amount of Ca in the rhizomorphs. The high concentration of K in S. granulatus rhizomorphs suggests that this fungus is a good accumulator of K while P. involutus appeared to accumulate heavy metals originating from wood ash.