In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi.

Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.

Uptake of vanadium and its intracellular metabolism by Coprinellus truncorum mycelial biomass.

BACKGROUND: Fungi absorb and solubilize a broad spectrum of heavy metals such as vanadium (V), which makes them a main route of its entry into the biosphere. V as vanadate (V5+) is a potential medical agent due to its many metabolic actions such as interaction with phosphates in the cell, and especially its insulin-mimetic activity. Antidiabetic activity of V-enriched fungi has been studied in recent years, but the biological and chemical bases of vanadium action and status in fungi in general are poorly understood, with almost no information on edible fungi. METHODS: This manuscript gives a deeper insight into the interaction of V5+ with Coprinellus truncorum, an edible autochthonous species widely distributed in Europe and North America. Vanadium uptake and accumulation as V5+ was studied by 51V NMR, while the reducing abilities of the mycelium were determined by EPR. 31P NMR was used to determine its effects on the metabolism of phosphate compounds, with particular focus on phosphate sugars identified using HPLC. RESULTS: Vanadate enters the mycelium in monomeric form and shows no immediate detrimental effects on intracellular pH or polyphosphate (PPc) levels, even when applied at physiologically high concentrations (20 mM Na3VO4). Once absorbed, it is partially reduced to less toxic vanadyl (V4+) with notable unreduced portion, which leads to a large increase in phosphorylated sugar levels, especially glucose-1-phosphate (G1P) and fructose-6-phosphate (F6P). CONCLUSIONS: Preservation of pH and especially PPc reflects maintenance of the energy status of the mycelium, i.e., its tolerance to high V5+ concentrations. Rise in G1P and F6P levels implies that the main targets of V5+ are most likely phosphoglucomutase and phosphoglucokinase(s), enzymes involved in early stages of G6P transformation in glycolysis and glycogen metabolism. This study recommends C. truncorum for further investigation as a potential antidiabetic agent.

ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore.

Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP, added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τin, and slower decline of τin during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi.

Osmotically Activated Anion Current of Phycomyces Blakesleeanus-Filamentous Fungi Counterpart to Vertebrate Volume Regulated Anion Current.

Studies of ion currents in filamentous fungi are a prerequisite for forming a complete understanding of their physiology. Cytoplasmic droplets (CDs), obtained from sporangiophores of Phycomyces blakesleeanus, are a model system that enables the characterization of ion currents in the native membrane, including the currents mediated by the channels not yet molecularly identified. Osmotically activated anionic current with outward rectification (ORIC) is a dominant current in the membrane of cytoplasmic droplets under the conditions of hypoosmotic stimulation. We have previously reported remarkable functional similarities of ORIC with the vertebrate volume regulated anionic current (VRAC), such as dose-dependent activation by osmotic difference, ion selectivity sequence, and time and voltage dependent profile of the current. Using the patch clamp method on the CD membrane, we further resolve VRAC-like ORIC characteristics in this paper. We examine the inhibition by extracellular ATP and carbenoxolone, the permeation of glutamate in presence of chloride, selectivity for nitrates, and activation by GTP, and we show its single channel behavior in excised membrane. We propose that ORIC is a functional counterpart of vertebrate VRAC in filamentous fungi, possibly with a similar essential role in anion efflux during cell volume regulation.

Cytotoxic activity of the crude polysaccharides/exopolysaccharides of Coprinus comatus and Coprinellus truncorum.

Fungi are an important source of polysaccharides (PSH) and phenolic compounds (PC). Numerous studies have highlighted the beneficial effects of fungal consumption, but the impact of submerged cultivated mycelia (M) and filtrate (F) has not been fully investigated. We aimed to investigate the cytotoxic activity of isolated crude PSH and exopolysaccharides (ePSH) of submerged cultivated M and F of edible Coprinus comatus and Coprinellus truncorum species. Both PSH and ePSH exhibited significant cytotoxic activity towards HepG2 cancer cells of human origin (three-way ANOVA). The C. truncorum PSH/ePSH was more efficient inducing maximal reduction in cell viability (≈50% at 450 µg/mL) after 24 h while C. comatus PSH/ePSH needed 72 h to reach similar effect (≈60% at 450 µg/mL). Partial least square regression (PLSR) analysis indicated that specific phenolic composition of the PSH/ePSH could be responsible for the difference in their activity.

Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi.

We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus. THG microscopy images showed bright spherical features dispersed throughout the hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. Colocalization analysis of THG and lipid-counterstained images disclosed that the cytoplasmic particles were lipid droplets. Particle Size Analysis and Image Correlation Spectroscopy were used to quantify the number density and size of lipid droplets. The two analysis methods both revealed an increase from 16 × 10-3 to 23 × 10-3 lipid droplets/µm2 after nitrogen starvation and a decrease in the average size of the droplets (range: 0.5-0.8 µm diameter). In conclusion, THG imaging, followed by PSA and ICS, can be reliably used for filamentous fungi for the in vivo quantification of lipid droplets without the need for labeling and/or fixation. In addition, it has been demonstrated that ICS is suitable for THG microscopy.

Biotransformation of selenium in the mycelium of the fungus Phycomyces blakesleeanus.

Biotransformation of toxic selenium ions to non-toxic species has been mainly focused on biofortification of microorganisms and production of selenium nanoparticles (SeNPs), while far less attention is paid to the mechanisms of transformation. In this study, we applied a combination of analytical techniques with the aim of characterizing the SeNPs themselves as well as monitoring the course of selenium transformation in the mycelium of the fungus Phycomyces blakesleeanus. Red coloration and pungent odor that appeared after only a few hours of incubation with 10 mM Se+4 indicate the formation of SeNPs and volatile methylated selenium compounds. SEM-EDS confirmed pure selenium NPs with an average diameter of 57 nm, which indicates potentially very good medical, optical, and photoelectric characteristics. XANES of mycelium revealed concentration-dependent mechanisms of reduction, where 0.5 mM Se+4 led to the predominant formation of Se-S-containing organic molecules, while 10 mM Se+4 induced production of biomethylated selenide (Se-2) in the form of volatile dimethylselenide (DMSe) and selenium nanoparticles (SeNPs), with the SeNPs/DMSe ratio rising with incubation time. Several structural forms of elemental selenium, predominantly monoclinic Se8 chains, together with trigonal Se polymer chain, Se8 and Se6 ring structures, were detected by Raman spectroscopy.

Synthesis and Biological Screening of New 4-Hydroxycoumarin Derivatives and Their Palladium(II) Complexes.

Two newly synthesized 4-hydroxycoumarin bidentate ligands (L1 and L2) and their palladium(II) complexes (C1 and C2) were screened for their biological activities, in vitro and in vivo. Structures of new compounds were established based on elemental analysis, 1H NMR, 13C NMR, and IR spectroscopic techniques. The obtained compounds were tested for their antioxidative and cytotoxic activities and results pointed to selective antiradical activity of palladium(II) complexes towards •OH and -•OOH radicals and anti-ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical) activity comparable to that of ascorbate. Results indicated the effect of C1 and C2 on the enzymatic activity of the antioxidative defense system. In vitro cytotoxicity assay performed on different carcinoma cell lines (HCT166, A375, and MIA PaCa-2), and one healthy fibroblast cell line (MRC-5) showed a cytotoxic effect of both C1 and C2, expressed as a decrease in carcinoma cells' viability, mostly by induction of apoptosis. In vivo toxicity tests performed on zebrafish embryos indicated different effects of C1 and C2, ranging from adverse developmental effect to no toxicity, depending on tested concentration. According to docking studies, both complexes (C1 and C2) showed better inhibitory activity in comparison to other palladium(II) complexes.

Biochemical and histological characterization of succulent plant Tacitus bellus response to Fusarium verticillioides infection in vitro.

We present changes in Tacitus bellus antioxidative system that specifically correspond to subsequent phases of hemibiotroph Fusarium verticillioides infection revealed by histological analysis. T. bellus response to spore germination 6 h post inoculation (hpi), manifested as first oxidative burst, was characterized by transient decrease in malondialdehyde (MDA) content, transient increase in catalase (CAT), low level of superoxide dismutase (SOD) and peroxidase (POD) activity, as well as with transient decrease in total antioxidant capacity (TAC), total phenol content (TPC) and phenylalanine ammonium lyase activity (PAL), and no changes in polyphenol oxidase (PPO) activity, or phenolic profile. During the biotrophic phase of F. verticillioides infection, characterized by hyphae spread intercellularly in epidermal and mesophyll tissue, the host antioxidative system was suppressed. The transition to necrotrophic phase of F. verticillioides infection (inter- and intracellular colonization and sporulation), occurred 3-4 days post inoculation (dpi). During the necrotrophic phase, 5-7 dpi, slowed progression of colonization of T. bellus mesophyll cells occurred and it coincided with sharp increase in MDA content and CAT, SOD and POD activities, but the drop in TAC, TPC content, and PPO activity, as well as the production of phytotoxin fusaric acid. Presented results add to the knowledge of events and mechanisms related to the transition from biotrophy to necrotrophy in F. verticillioides.

Fatty Acids Predominantly Affect Anti-Hydroxyl Radical Activity and FRAP Value: The Case Study of Two Edible Mushrooms.

Compared to plants, nowadays mushrooms attract more attention as functional foods, due to a number of advantages in manipulating them. This study aimed to screen the chemical composition (fatty acids and phenolics) and antioxidant potential (OH•, 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and ferric reducing ability of plasma (FRAP)) of two edible mushrooms, Coprinus comatus and Coprinellus truncorum, collected from nature and submerged cultivation. Partial least square regression analysis has pointed out the importance of some fatty acids-more precisely, unsaturated fatty acids (UFAs) followed by fatty acids possessing both short (C6:0 and C8:0) and long (C23:0 and C24:0) saturated chains-and phenolic compounds (such as protocatechuic acid, daidzein, p-hydroxybenzoic acid, genistein and vanillic acid) for promising anti-OH•, FRAP and anti-DPPH• activities, respectively. However, other fatty acids (C16:0, C18:0 and C18:3n3) along with the flavonol isorhamnetin are actually suspected to negatively affect (by acting pro-oxidative) the aforementioned parameters, respectively. Taken together, design of new food supplements targeting oxidative stress might be predominantly based on the various UFAs combinations (C18:2n6, C20:1, C20:2, C20:4n6, C22:2, C22:1n9, etc.), particularly if OH• is suspected to play an important role.

The response of chironomid taxonomy- and functional trait-based metrics to fish farm effluent pollution in lotic systems.

The lotic habitats affected by trout farm waste are colonized with a particular invertebrate community of which chironomids are the most abundant group. However, there is little information available regarding how chironomid community structures respond to this type of pollution at the highest taxonomic resolution. Eight fish farms, together with their lotic systems as recipients, were used to test the variability of the chironomid community and its surrogates (taxonomic and functional metrics) across spatially arranged sampling sites to form a gradual decrease in the trout farm influence. The self organizing map (SOM) classified six different types of chironomid communities which were characteristic for both the control and affected habitats. The species indicator analyses listed 32 taxa as positive indicators of water pollution. The SOM and Kruskal-Wallis test revealed that the pattern of chironomid community structure obtained was mainly driven by six environmental parameters (Altitude, conductivity, distance from the outlet, hardness, HN4-N, NO3-N). Categorical principal components analysis (CATPCA) derived three models for each type of biotic metric, in which for diversity-, taxonomy- and functional feeding group-based metrics, the first two dimensions explained 55.2%, 58.3% and 55.4%, of the total variance respectively for 315 sampling sites. According to this analysis, the total number of taxa (S), abundance and the Shannon-Wiener index (H') (as a diversity metric), as well as the proportion of Tanypodinae (as taxonomic group) and grazers/scraper (GRA) and gatherer collector (GAT)(as FFG metrics), were related to the outlet distance gradient, thus showing great potential to be used in the multimetric approach in bioassessment.

Evaluation of oxidative stress biomarkers in the freshwater gammarid Gammarus dulensis exposed to trout farm outputs.

The current study reports a combined seasonal monitoring of the Crnica River watercourse using physico-chemical parameters and biomarkers measured in Gammarus dulensis with the aim of correlating the effects of pollution with the level of oxidative stress biomarkers in this amphipod. The pollution source is identified as a trout farm in the upper part of the Crnica River (Eastern Serbia). Physico-chemical water parameters and sediment trace element concentrations were measured in the Crnica River during three seasons. Three localities downstream [CR2, CR3, and CR4 (20 m, 400 m, and 1.3 km from the trout farm discharge, respectively)] were assessed in comparison with a reference location (CR1), 250 m upstream from the trout aquaculture in the Crnica River. Antioxidant biomarkers including the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione-S-transferase (GST) were determined in pooled samples of G. dulensis. Our findings showed statistically different antioxidant enzyme levels in gammarids from the first downstream locality (CR2) compared to those from other three localities. The results indicate that oxidative stress occurred to some extent despite the assumed antioxidant induction as a result of exposure to elevated levels of some elements and changed water parameters, the effects seeming in part to be mediated by suppression of SOD and CAT (during summer and autumn); and by suppression of GPX and GR (during autumn) at the CR2 locality. The activity of GST in all seasons was higher at the CR2 locality than at the other localities, suggesting the presence of some permanent xenobiotic next to the aquaculture output. Seasonal difference in the physical and chemical water parameters and sediment trace element concentrations were observed. Overall, our results indicate a locality- and season-dependent antioxidant enzymes response in G. dulensis, pointing at progressively reduced deleterious effect of trout farm outputs with movement further downstream.

Can Oxidation-Reduction Potential of Cerebrospinal Fluid Be a Monitoring Biomarker in Amyotrophic Lateral Sclerosis?

The monitoring of progression in amyotrophic lateral sclerosis (ALS) relies on clinical outcome measures that take months to interpret, such as revised ALS functional rating scale (ALSFRS-R) score, with no approved biomarkers. A number of clinical studies have documented the involvement of oxidative stress in ALS pathology. Pertinent to this, we propose to evaluate oxidation-reduction potential (ORP) of cerebrospinal fluid (CSF) as a potential indicator of ALS progression. The case-control study included 24 patients with neurological non-neurodegenerative disorders (controls) and 82 ALS patients with different degrees of disease (ALSFRS-R score: 21-47). ORP was significantly higher in ALS patients than controls. It was not dependent on age or gender. A strong negative correlation was found between ORP and ALSFRS-R score for all patients and patients with spinal onset. In other words, ORP increased with ALS progression. No correlation was found for the subset of patients with bulbar onset, most likely because of the physical distance between neurodegenerative loci and the site of CSF collection. These results lead to the hypothesis that ORP of CSF has a potential as monitoring biomarker in ALS, particularly in the cohort of patients with spinal onset. Antioxid. Redox Signal. 28, 1570-1575.

Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites.

Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 µM NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33±5 and 21±3 % of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.

The interactions of vanadate monomer with the mycelium of fungus Phycomyces blakesleeanus: reduction or uptake?

The possibility of reduction of vanadate monomer in the mycelium of fungus Phycomyces blakesleeanus was investigated in this study by means of polarography. Control experiments were performed with vanadyl [V(IV)] and vanadate [V(V)] in 10 mM Hepes, pH 7.2. Addition of P. blakesleeanus mycelium resulted in disappearance of all V(IV) polarographic waves recorded in the control. This points to the uptake of all available V(IV) by the mycelium, up to 185 µmol/gFW, and suggests P. blakesleeanus as a potential agent in V(IV) bioremediation. Polarographic measurements of mycelium with low concentrations (0.1-1 mM) of V(V), that only allows the presence of monomer, showed that fungal mycelia removes around 27% of V(V) from the extracellular solution. Uptake was saturated at 104 ± 2 µmol/gFW which indicates excellent bioaccumulation capability of P. blakesleeanus. EPR, 51V NMR and polarographic experiments showed no indications of any measurable extracellular complexation of V(V) monomer with fungal exudates, reduction by the mycelium or adsorption to the cell wall. Therefore, in contrast to vanadium oligomers, vanadate monomer interactions with the mycelium are restricted to its transport into the fungal cell, probably by a phosphate transporter.

(51)V NMR investigation of cell-associated vanadate species in Phycomyces blakesleeanus mycelium.

(51)V NMR spectroscopy was used for detection and identification of cell-associated vanadate (V(5+)) species after exposure of Phycomyces blakesleeanus mycelium, in exponential phase of growth, to sodium orthovanadate. Complete disappearance of monomer and dimer signals and decreased intensity of the tetramer signal were observed about 40 min after treatment. Simultaneously, a signal at -532 ppm, with increasing intensity, was detected in spectra. The time-dependent rise in this signal was connected to a decrease in the extracellular monomer signal, indicating its transport into the cell. The signal at -532 ppm did not belong to any known simple oxido-vanadate species, nor to a complex with any of the components of experimental medium. This signal was the only one present in spectrum of the mycelium washed 35 min after treatment, and the only one observed in mycelium cultivated on vanadate-contained medium. Therefore, its appearance can be attributed to intracellular complexation, and may represent an important detoxification mechanism of the cell exposed to a physiologically relevant concentration of vanadate. Experiments ((51)V NMR and polarography) performed with Cd-pretreated mycelium (inhibitor of an enzyme responsible for V(5+) reduction) and ferricyanide-preincubated mycelium excluded the possibility of V(5+) tetramer's entry into the cell.

Osmotic swelling activates a novel anionic current with VRAC-like properties in a cytoplasmic droplet membrane from Phycomyces blakesleeanus sporangiophores.

We describe here whole-cell currents of droplets prepared from the apical region of growing Phycomyces blakesleeanus sporangiophores. Whole-cell current recordings revealed the osmotically activated, outwardly rectifying, fast inactivating instantaneous current (ORIC) with biophysical properties closely resembling volume-regulated anionic current (VRAC). ORIC is activated under conditions of osmotically induced swelling and shows strong selectivity for anions over cations. In addition, ORIC shows voltage and time-dependent inactivation at positive potentials and recovery from inactivation at negative potentials. ORIC is blocked by anthracene-9-carboxylic acid, an anion channel blocker, in a voltage-dependent manner. This is the first report of the presence of VRAC-like current in an organism outside the chordate lineage.

Vanadate influence on metabolism of sugar phosphates in fungus Phycomyces blakesleeanus.

The biological and chemical basis of vanadium action in fungi is relatively poorly understood. In the present study, we investigate the influence of vanadate (V5+) on phosphate metabolism of Phycomyces blakesleeanus. Addition of V5+ caused increase of sugar phosphates signal intensities in 31P NMR spectra in vivo. HPLC analysis of mycelial phosphate extracts demonstrated increased concentrations of glucose 6 phosphate, fructose 6 phosphate, fructose 1, 6 phosphate and glucose 1 phosphate after V5+ treatment. Influence of V5+ on the levels of fructose 2, 6 phosphate, glucosamine 6 phosphate and glucose 1, 6 phosphate (HPLC), and polyphosphates, UDPG and ATP (31P NMR) was also established. Increase of sugar phosphates content was not observed after addition of vanadyl (V4+), indicating that only vanadate influences its metabolism. Obtained results from in vivo experiments indicate catalytic/inhibitory vanadate action on enzymes involved in reactions of glycolysis and glycogenesis i.e., phosphoglucomutase, phosphofructokinase and glycogen phosphorylase in filamentous fungi.

Oxygen regulation of alternative respiration in fungus Phycomyces blakesleeanus: connection with phosphate metabolism.

Environmental changes can often result in oxygen deficiency which influences cellular energy metabolism, but such effects have been insufficiently studied in fungi. The effects of oxygen deprivation on respiration and phosphate metabolites in Phycomyces blakesleeanus were investigated by oxygen electrode and (31)P NMR spectroscopy. Mycelium was incubated in hypoxic and anoxic conditions for 1.5, 3 and 5 h and then reoxygenated. Participation of alternative oxidase (AOX) in total respiration increased gradually in both treatments and after 5 h of anoxia exceeded a value 50% higher than in control. Shortly after reintroduction of oxygen into the system AOX level decreased close to the control level. Oxygen deprivation also caused a reversible decrease of polyphosphate/inorganic phosphate ratio (PPc/Pi), which was strongly correlated with the increase of AOX participation in total respiration. Unexpectedly, ATP content remained almost constant, probably due to the ability of PolyP to sustain energy and phosphate homeostasis of the cell under stress conditions. This was further substantiated by the effects of azide, a cytochrome c oxidase inhibitor, which also decreased PPc/Pi ratio, but to a smaller extent in oxygen deprived than control and reoxygenated specimens.

The interactions of vanadium with Phycomyces blakesleeanus mycelium: enzymatic reduction, transport and metabolic effects.

The biological and chemical basis of vanadium action and transport in fungi is relatively poorly understood. In this study we investigated the interactions of vanadium in physiologically-relevant redox states: vanadate (+5) and vanadyl (+4), with mycelium of fungus Phycomyces blakesleeanus using EPR and (31)P NMR spectroscopy and biochemical assays. We determined that P. blakesleeanus reduces V(5+) to V(4+) in the extracellular compartment by the means of cell surface enzyme with ferricyanide reductase activity, which contains molybdenum-molybdopterin as a cofactor. Both, V(5+) and V(4+) bind to cell wall. They enter the cytoplasm via phosphate transporter and cation channels, respectively, and exhibit different metabolic effects. Vanadate provokes increased biomass production, the effects being inverted to toxic at higher V(5+) concentrations. In addition, V(5+) activates the synthesis of sugar phosphates and oligophosphates. On the other hand, V(4+) exhibits toxic effects even at low concentrations. The V(4+) detoxification route involves binding to vacuolar polyphosphates. Altogether our results imply that the mechanism of interaction of vanadium with P. blakesleeanus involves three major steps: extracellular enzymatic V(5+)/V(4+) reduction, V(4+) influx, and vacuolar storage, with an additional step - V(5+) import occurring at higher vanadate concentrations.