Long-term effects of oil contamination on soil quality and metabolic function.

Widespread soil contamination with oil and the toxicity of petroleum hydrocarbons to soil biota make it extremely important to study microbial responses to oil stress. Soil metabolites reflect the main metabolic pathways in the soil microbial community. The examination of changes in the soil metabolic profile and metabolic function is essential for a better understanding of the nature of the pollution and restoration of the disturbed soils. The present study aimed to assess the long-term effect of oil on the ecological state of the soil, evaluate quantitative and qualitative differences in metabolite composition between soil contaminated with oil and non-contaminated soil, and reveal biologically active metabolites that are related to oil contamination and can be used for contamination assessment. A long-term field experiment was conducted to examine the effects of various oil concentrations on the biochemical properties and metabolic profile of the soil. Podzolic soil contaminated with oil demonstrated the long-term inhibition of soil biological activity and vegetation. Oil affected the metabolic activity of soil fungi increasing the production of toxic metabolites. A metabolomic approach was employed to determine soil metabolites. The metabolite profile was found to vary greatly between oil-contaminated and non-contaminated soils. Carbohydrates had the largest number of metabolites negatively affected by oil, while the content of organic acids, phenolic compounds, and terpenoids was mainly increased in oil-contaminated soil. The evaluation of the long-term impact of oil on microbial metabolism can make a valuable contribution to the assessment of soil quality and the activity of soil microorganisms being under stress from oil pollution. The results contribute to a further understanding of the role of microorganisms in the ecological functions of contaminated soil, which can be useful in the development of rehabilitation strategies for disturbed sites.

The Role of Medicago lupulina Interaction with Rhizophagus irregularis in the Determination of Root Metabolome at Early Stages of AM Symbiosis.

The nature of plant-fungi interaction at early stages of arbuscular mycorrhiza (AM) development is still a puzzling problem. To investigate the processes behind this interaction, we used the Medicago lupulina MlS-1 line that forms high-efficient AM symbiosis with Rhizophagus irregularis. AM fungus actively colonizes the root system of the host plant and contributes to the formation of effective AM as characterized by a high mycorrhizal growth response (MGR) in the host plant. The present study is aimed at distinguishing the alterations in the M. lupulina root metabolic profile as an indicative marker of effective symbiosis. We examined the root metabolome at the 14th and 24th day after sowing and inoculation (DAS) with low substrate phosphorus levels. A GS-MS analysis detected 316 metabolites. Results indicated that profiles of M. lupulina root metabolites differed from those in leaves previously detected. The roots contained fewer sugars and organic acids. Hence, compounds supporting the growth of mycorrhizal fungus (especially amino acids, specific lipids, and carbohydrates) accumulated, and their presence coincided with intensive development of AM structures. Mycorrhization determined the root metabolite profile to a greater extent than host plant development. The obtained data highlight the importance of active plant-fungi metabolic interaction at early stages of host plant development for the determination of symbiotic efficiency.

Contribution to Studies on Rare Medicinal Mushroom Sparassis crispa (Agaricomycetes) in Culture and Its Production of Phenol Compounds under Various Cultivation Conditions.

Three Sparassis crispa strains from the Komarov Botanical Institute Basidiomycetes Culture Collection (LE-BIN) were studied on various agar and liquid media for growth and phenol compound production. On agar media, the strains produced crystals of various shapes and sizes that glowed in ultraviolet light and were visible to the naked eye. Using gas chromatography-mass spectrometry, the crystals were identified as sparassol (methyl 2-hydroxy-4-methoxy-6-methylbenzoate). Other phenol compounds (i.e., methyl ester of sparassol and methyl ester of orsellinic acid) were also found in S. crispa mycelium. Phenol compounds were detected both in the mycelium and culture filtrate of all studied strains cultivated on various agar and liquid media, at different acidity and duration, but their composition and ratio varied. The initial pH of cultivated medium did not have a large influence on phenol compound production, unlike culture growth duration, which correlated directly with sparassol concentration in mycelium. Production of the phenol compounds presumably connected with constitutive processes of Sparassis metabolism, but the intensity of their production and accumulation in mycelium depends on the strain, conditions, and cultivation time. Strain LE-BIN 2902 is considered a promising sparassol producer.

Mycorrhiza-Induced Alterations in Metabolome of Medicago lupulina Leaves during Symbiosis Development.

The present study is aimed at disclosing metabolic profile alterations in the leaves of the Medicago lupulina MlS-1 line that result from high-efficiency arbuscular mycorrhiza (AM) symbiosis formed with Rhizophagus irregularis under condition of a low phosphorus level in the substrate. A highly effective AM symbiosis was established in the period from the stooling to the shoot branching initiation stage (the efficiency in stem height exceeded 200%). Mycorrhization led to a more intensive accumulation of phosphates (glycerophosphoglycerol and inorganic phosphate) in M. lupulina leaves. Metabolic spectra were detected with GS-MS analysis. The application of complex mathematical analyses made it possible to identify the clustering of various groups of 320 metabolites and thus demonstrate the central importance of the carbohydrate and carboxylate-amino acid clusters. The results obtained indicate a delay in the metabolic development of mycorrhized plants. Thus, AM not only accelerates the transition between plant developmental stages but delays biochemical "maturation" mainly in the form of a lag of sugar accumulation in comparison with non-mycorrhized plants. Several methods of statistical modeling proved that, at least with respect to determining the metabolic status of host-plant leaves, stages of phenological development have priority over calendar age.

Comparative Analysis of Dental Pulp and Periodontal Stem Cells: Differences in Morphology, Functionality, Osteogenic Differentiation and Proteome.

Dental stem cells are heterogeneous in their properties. Despite their common origin from neural crest stem cells, they have different functional capacities and biological functions due to niche influence. In this study, we assessed the differences between dental pulp stem cells (DPSC) and periodontal ligament stem cells (PDLSC) in their pluripotency and neuroepithelial markers transcription, morphological and functional features, osteoblast/odontoblast differentiation and proteomic profile during osteogenic differentiation. The data were collected in paired observations: two cell cultures, DPSC and PDLSC, were obtained from each donor. Both populations had the mesenchymal stem cells surface marker set exposed on their membranes but differed in Nestin (a marker of neuroectodermal origin) expression, morphology, and proliferation rate. OCT4 mRNA was revealed in DPSC and PDLSC, while OCT4 protein was present in the nuclei of DPSC only. However, transcription of OCT4 mRNA was 1000-10,000-fold lower in dental stem cells than in blastocysts. DPSC proliferated at a slower rate and have a shape closer to polygonal but they responded better to osteogenic stimuli as compared to PDLSC. RUNX2 mRNA was detected by qPCR in both types of dental stem cells but RUNX2 protein was detected by LC-MS/MS shotgun proteomics only in PDLSC suggesting the posttranscriptional regulation. DSPP and DMP1, marker genes of odontoblastic type of osteogenic differentiation, were transcribed in DPSC but not in PDLSC samples. Our results prove that DPSC and PDLSC are different in their biology and therapeutic potential: DPSC are a good candidate for osteogenic or odontogenic bone-replacement cell-seeded medicines, while fast proliferating PDLSC are a prospective candidate for other cell products.

Fullerenol changes metabolite responses differently depending on the iron status of cucumber plants.

The unique properties of carbon-based nanomaterials, including fullerenol, have attracted great interest in agricultural and environmental applications. Iron (Fe) is an essential micronutrient for major metabolic processes, for which a shortage causes chlorosis and reduces the yield of many crops cultivated worldwide. In the current study, the metabolic responses of Cucumis sativus (a Strategy I plant) to fullerenol treatments were investigated depending on the Fe status of plants. Cucumber plants were grown hydroponically, either with [+FeII (ferrous) and +FeIII (ferric)] or in Fe-free (-FeII and -FeIII) nutrient solution, with (+F) or without (-F) a fullerenol supply. Iron species-dependent effects were observed in either Fe-fed or Fe-starved plants, with alteration of metabolites involved in the metabolism of carbohydrates, amino acids, organic acids, lipophilic compounds. Metabolic perturbations triggered by fullerenol in the FeIII-treated plants were in the opposite kind from those in the FeII-treated plants. Whereas in the FeIII-fed plants, fullerenol activated the metabolisation of carbohydrates and amino acids, in the FeII-fed plants, fullerenol activated the metabolisation of lipophilic compounds and repressed the metabolisation of carbohydrates and amino acids. In FeIII-deficient plants, fullerenol stimulated the metabolism of C3 carboxylates and lipophilic compounds while repressing the metabolism of amino acids, hexoses and dicarboxylates, while in FeII-deficient plants, activations of the metabolism of amino acids and dicarboxylates and repression of sterol metabolism by fullerenol were observed. The results indicated that the valence state of Fe sources is of importance for re-programming metabolome responses in cucumber to fullerenol either in Fe-sufficient or Fe-deficient conditions. These investigations are significant for understanding fullerenol interactions and risk assessment in plants with different Fe statuses.

Rhizosphere Bacterium Rhodococcus sp. P1Y Metabolizes Abscisic Acid to Form Dehydrovomifoliol.

The phytohormone abscisic acid (ABA) plays an important role in plant growth and in response to abiotic stress factors. At the same time, its accumulation in soil can negatively affect seed germination, inhibit root growth and increase plant sensitivity to pathogens. ABA is an inert compound resistant to spontaneous hydrolysis and its biological transformation is scarcely understood. Recently, the strain Rhodococcus sp. P1Y was described as a rhizosphere bacterium assimilating ABA as a sole carbon source in batch culture and affecting ABA concentrations in plant roots. In this work, the intermediate product of ABA decomposition by this bacterium was isolated and purified by preparative HPLC techniques. Proof that this compound belongs to ABA derivatives was carried out by measuring the molar radioactivity of the conversion products of this phytohormone labeled with tritium. The chemical structure of this compound was determined by instrumental techniques including high-resolution mass spectrometry, NMR spectrometry, FTIR and UV spectroscopies. As a result, the metabolite was identified as (4RS)-4-hydroxy-3,5,5-trimethyl-4-[(E)-3-oxobut-1-enyl]cyclohex-2-en-1-one (dehydrovomifoliol). Based on the data obtained, it was concluded that the pathway of bacterial degradation and assimilation of ABA begins with a gradual shortening of the acyl part of the molecule.

Lipid and Metabolite Profiling of Serpula lacrymans Under Freezing Stress.

Basidiomycete fungus Serpula lacrymans is one of the most dangerous indoor fungus causing dry rot of timber. The physiology of this fungus deserves more attention as a basis for development of methods of dry rot treatment. We observed an increase in the freezing resistance of S. lacrymans after pre-cultivation of mycelia at elevated temperatures. To examine the biochemical mechanisms underlying this phenomenon the lipid composition and metabolite profiling of mycelia subjected to freezing and thawing were investigated. An analysis is made of the growth rate and metabolism of "daughter" cultures derived from a frozen mycelia. According to the results, sphingolipids and water-soluble metabolites such as mannitol, glycerol, sugar alcohols, some amino- and organic acids are able to function as protective compounds providing a cross-resistance between heat shock and freeze-thaw stress in S. lacrymans.

Metabolic alterations in pea leaves during arbuscular mycorrhiza development.

Arbuscular mycorrhiza (AM) is known to be a mutually beneficial plant-fungal symbiosis; however, the effect of mycorrhization is heavily dependent on multiple biotic and abiotic factors. Therefore, for the proper employment of such plant-fungal symbiotic systems in agriculture, a detailed understanding of the molecular basis of the plant developmental response to mycorrhization is needed. The aim of this work was to uncover the physiological and metabolic alterations in pea (Pisum sativum L.) leaves associated with mycorrhization at key plant developmental stages. Plants of pea cv. Finale were grown in constant environmental conditions under phosphate deficiency. The plants were analyzed at six distinct time points, which corresponded to certain developmental stages of the pea: I: 7 days post inoculation (DPI) when the second leaf is fully unfolded with one pair of leaflets and a simple tendril; II: 21 DPI at first leaf with two pairs of leaflets and a complex tendril; III: 32 DPI when the floral bud is enclosed; IV: 42 DPI at the first open flower; V: 56 DPI when the pod is filled with green seeds; and VI: 90-110 DPI at the dry harvest stage. Inoculation with Rhizophagus irregularis had no effect on the fresh or dry shoot weight, the leaf photochemical activity, accumulation of chlorophyll a, b or carotenoids. However, at stage III (corresponding to the most active phase of mycorrhiza development), the number of internodes between cotyledons and the youngest completely developed leaf was lower in the inoculated plants than in those without inoculation. Moreover, inoculation extended the vegetation period of the host plants, and resulted in increase of the average dry weight per seed at stage VI. The leaf metabolome, as analyzed with GC-MS, included about three hundred distinct metabolites and showed a strong correlation with plant age, and, to a lesser extent, was influenced by mycorrhization. Metabolic shifts influenced the levels of sugars, amino acids and other intermediates of nitrogen and phosphorus metabolism. The use of unsupervised dimension reduction methods showed that (i) at stage II, the metabolite spectra of inoculated plants were similar to those of the control, and (ii) at stages IV and V, the leaf metabolic profiles of inoculated plants shifted towards the profiles of the control plants at earlier developmental stages. At stage IV the inoculated plants exhibited a higher level of metabolism of nitrogen, organic acids, and lipophilic compounds in comparison to control plants. Thus, mycorrhization led to the retardation of plant development, which was also associated with higher seed biomass accumulation in plants with an extended vegetation period. The symbiotic crosstalk between host plant and AM fungi leads to alterations in several biochemical pathways the details of which need to be elucidated in further studies.

Role of lipids in the thermal plasticity of basidial fungus Favolaschia manipularis.

In this study, we examined the lipid composition of two strains of the tropical basidiomycete Favolaschia manipularis (Berk.) Teng, which differ in their adaptive potential to high (35 °C) and low (5 °C) temperatures. The results suggest that adaptation to extreme temperatures involves a change in the molecular composition of sterols, in addition to other well-known mechanisms of regulating membrane thickness and fluidity, such as changes in the lipid unsaturation and in the proportion of bilayer- and non-bilayer-forming lipids. It was demonstrated for the first time that adaptation to high temperature stress in fungi is accompanied by the accumulation of 9(11)-dehydroergosterol and ergosterol peroxide. Furthermore, increased thermal plasticity correlates with high storage lipid (triglycerides) content, accumulation of phosphatidic acid in the membrane, and an equal proportion of bilayer and non-bilayer lipids in the membrane.

Mimicking pollen and spore walls: self-assembly in action.

BACKGROUND AND AIMS: Decades of research have attempted to elucidate the underlying developmental mechanisms that give rise to the enormous diversity of pollen and spore exines. The organization of the exine starts with the establishment of an elaborate glycocalyx within which the subsequent accumulation of sporopollenin occurs. Ontogenetic studies using transmission electron microscopy of over 30 species from many different groups have shown that the sequence of structures observed during development of the exine corresponds to the sequence of self-assembling micellar mesophases (including liquid crystals) observed at increasing concentrations of surfactants. This suggested that self-assembly plays an important part in exine pattern determination. Some patterns resembling separate layers of spore and pollen grain walls have been obtained experimentally, in vitro, by self-assembly. However, to firmly establish this idea, columellate and granulate exines, the most widespread forms, needed to be simulated experimentally. METHODS: We used our original method, preparing mixtures of substances analogous to those known to occur in the periplasmic space of developing microspores, then leaving the mixtures undisturbed for specific periods of time to allow the process of self-assembly to occur. We developed our method further by using new substances analogous to those present in the periplasmic space and performing the experiments in a thin layer, more closely resembling the dimensions of the periplasmic space. KEY RESULTS: The artificial microstructures obtained from our in vitro self-assembly experiments closely resembled the main types of exines, including tectate-columellate, granulate, alveolate and structureless, and permitted comparison with both developing and mature microspore walls. Compared with the previous attempts, we managed to simulate columellate and granulate exines, including lamellate endexine. CONCLUSIONS: Our results show that simple physico-chemical interactions are able to generate patterns resembling those found in exines, supporting the idea that exine development in nature involves an interplay between the genome and self-assembly.

Silicon ameliorates iron deficiency of cucumber in a pH-dependent manner.

Strategy I plants may respond to iron (Fe) deficiency by rhizosphere acidification. Here, the role of medium pH-values in silicon (Si)-induced mitigation Fe deficiency in Strategy I plants (Cucumis sativus) was investigated, particularly the metabolites regulated by a lack of Fe, using a target metabolomics approach. Plants were grown hydroponically, either with (+Fe) or in Fe-free (-Fe) nutrient solution, with (+Si) or without (-Si) a Si supply. The nutrient solution was adjusted to pH 5.0 or 6.0 and checked daily. Leaf metabolites potentially involved in Fe transport were determined. The typical Fe responses of cucumber (e.g., decrease in leaf chlorophyll, Fe imbalance) were more pronounced when plants were grown at pH 6.0 than 5.0, during long-term Fe deficiency (15 days). Major metabolites up-regulated by Fe deficiency and found in young leaf were succinic, citric and glutamic acids, respectively; their maximal concentrations occurred in Fe-starved plants grown at pH 6.0 without Si supply. Silicon (Si)-induced effects accompanied with alleviation chlorosis symptoms, were most distinct in plants grown at pH 6.0 for an extended period without Fe. Changes in abundance of metabolites specifically up-regulated by a lack of Fe may be manifested before any Si-induced changes in plant Fe content were apparent, suggesting that metabolite responses are highly sensitive to a Fe-dependent signal altered by Si treatments under Fe deficiency. The results indicate that Si supply was more evident when plants were more stressed by an increase in nutrient solution pH under Fe-limited conditions.

Cultural and Metabolomic Studies of a New Phtalides Producer, Lignomyces vetlinianus (Agaricomycetes).

Culture characteristics and metabolomic profiling (on the basis of gas chromatography-mass spectrometry) of 3 strains of Lignomyces vetlinianus were studied. Growth rate, macromorphology, and micromorphology of mycelia grown on various media are described. More than 60 compounds were detected in the mycelial extracts, including amino acids, organic acids that are active during the tricarboxylic acid cycle, sugars, fatty acids, sugar alcohols, and sugar acids. Principal component analysis of low-molecular-weight compounds in mycelial methanol extracts of L. vetlinianus strains at different stages of growth demonstrated that the pattern of mycelial metabolomes grouped by age of the culture indicates a significant relation between the development of the culture and the specificity of its metabolite spectrum. Slow-growing cultures develop gradually and are characterized by several changes in metabolite states. The pattern of points is grouped more tightly for fast-growing strains. The production of crystal-like aggregates was observed for aging mycelia at the stationary phase of growth. These aggregates were isolated from mycelia and identified as clusters of 4,6-dimethoxy-phthalide. The molecular structure of this substance was confirmed by nuclear magnetic resonance analysis. The results show that the concentration of 4,6-dimethoxy-phthalide increased during cultivation. Fruiting bodies contained very small amounts of 4,6-dimethoxy-phthalide compared with amounts in mycelia. It can be assumed that L. vetlinianus is a powerful natural producer of phthalides of biotech-nological interest and can be used as a model to study phenolic metabolism in fungi.

Stomata control is changed in a chlorophyll b-free barley mutant.

The barley (Hordeum vulgare L.) chlorina f2 3613 mutant exhibits low photosynthesis and slow growth. This results from downregulation of the levels of photosynthetic antenna proteins caused by the absence of chl b, the major regulator of photosynthetic antennae in land plants. Here, we demonstrate that, when grown in the field in full sunlight, this mutant displays a changed pattern of stomatal responses compared with the parental wild-type cultivar Donaria. However, stomatal regulation of chlorina f2 3613 plants was restored when plants were placed under a shade cover for several days. The shade cover reduced incident PAR from 2000-2200µmolm-2s-1 to 800-880µmolm-2s-1 as measured at noon. Contents of ABA, the xanthophyll precursors of ABA biosynthesis and minor antenna proteins, as well as reactive oxygen species levels in stomata and the sensitivity of stomata to exogenously supplied ABA, were determined in leaves of wild-type Donaria and chlorina f2 3613 before and after shading. The results support the view that the restoration of stomatal control in barley chlorina f2 3613 is correlated with an increase in the levels of the minor antenna protein Lhcb6, which has recently been implicated in the enhancement of stomatal sensitivity to ABA in Arabidopsis thaliana (L.) Heynh.

Interactions between aluminium, iron and silicon in Cucumber sativus L. grown under acidic conditions.

Aluminium (Al) is one of the major stressors for plants in acidic soils, negatively affecting plant growth and nutrient balances. Significant efforts have been undertaken to understand mechanisms of Al tolerance in plants. However, little is known of the relevance of iron (Fe) and silicon (Si) nutrition under Al stress conditions. The objectives of this study were to determine whether effects induced by Fe and Si are of importance for limitation of Al moving via xylem in plants (Cucumis sativus L.). Cucumber plants (cv. Phoenix and Solovei) were grown (i) hydroponically in a complete nutrient solution at pH 4.0, either with (+Fe) or in Fe-free (-Fe) nutrient solution, without (-Si) or with (+Si) supply of Si, without (-Al) or with (+Al) exposure of Al and (ii) in soil. Xylem sap concentrations of Al, Fe and Si were measured. To characterise the pattern of xylem sap transport of Al and Fe, metabolomic changes of root tissues were investigated. Although the growth of cucumber plants was not significantly affected by Al3+ (Al-tolerant), Al exposure decreased xylem sap Fe (+Fe plants) and increased ferric chelate reductase (FC-R) activity of roots (-Fe plants). On the other hand, Fe supply greatly mitigated the Al-induced increase in xylem sap Al. The ameliorative effect of Fe depended on plant genotypes and was more pronounced in the more Fe-efficient cultivar Phoenix, which presented the highest level of xylem sap Fe. Xylem sap Fe was positively correlated with root serine, succinic and fumaric acids, suggesting that a probable underlying mechanism of Al tolerance might involve the chelation of Fe by biosynthesis of these chelating compounds. The Si-modulated root succinate increase appears to be of great importance for facilitating long-distance transport of Fe, thereby hindering Al transport from roots to shoots. The results highlight for the first time the importance of both Fe and Si supply in plant exclusion of Al under acidic conditions.

Novel brominated metabolites from Bryozoa: a functional analysis.

Marine invertebrates are a promising source of novel natural products with biological activities. The phylum Bryozoa is relatively under-investigated in this context, although a number of compounds with medical potential has been discovered in recent years. Here, we report on the novel group of brominated metabolites from the bryozoan Terminoflustra membranaceatruncata, including analysis of biological activities of the tribrominated terminoflustrindole A (Cm-1) and the structures of the related dibrominated variants terminoflustrindoles B and C. Terminoflustrindole A was previously shown to have fungicidal properties. Although they vary by just one bromine group in each case from terminoflustrindole A, in this study, we report that terminoflustrindoles B and C exhibit no antimicrobial activity in the same assays. In addition to displaying antifungal activity, Terminoflustrindole A was also found to exhibit potent cytotoxic activity when tested against tumour cell lines. The gradient distribution of this compound within the bryozoan colony was demonstrated using LC-MS-analysis.

Glandular trichomes of Tussilago Farfara (Senecioneae, Asteraceae).

MAIN CONCLUSION: The glandular trichomes are developed on the aerial organs of Tussilago farfara ; they produce phenols and terpenoids. Smooth endoplasmic reticulum and leucoplasts are the main organelles of the trichome secretory cells. The aim of this study was to characterise the morphology, anatomy, histochemistry and ultrastructure of the trichomes in Tussilago farfara as well as to identify composition of the secretory products. Structure of trichomes located on the peduncles, bracts, phyllaries, and leaves were studied by light and electron microscopy. The capitate glandular trichomes consist of a multicellular head and a biseriate long stalk. Histochemical tests and fluorescence microscopy reveal phenols and terpenoids in the head cells. During secretory stage, the head cells contain smooth and rough endoplasmic reticulum, Golgi apparatus, diversiform leucoplasts with opaque contents in lamellae, chloroplasts, mitochondria, and microbodies. In the capitate glandular trichomes of T. farfara subcuticular cavity is absent, unlike glandular trichomes in other Asteraceae species. For the first time, content of metabolites in the different vegetative and reproductive organs as well as in the isolated capitate glandular trichomes was identified by GC-MS. Forty-five compounds, including organic acids, sugars, polyols, phenolics, and terpenoids were identified. It appeared that metabolite content in the methanol extracts from peduncles, bracts and phyllaries is biochemically analogous, and similar to the metabolites from leaves, in which photosynthesis happens. At the same time, the metabolites from trichome extracts essentially differ and refer to the above-mentioned secondary substances. The study has shown that the practical value of the aerial organs of coltsfoot is provided with flavonoids produced in the capitate glandular trichomes.

Properties of substances inhibiting aggregation of oxidized GAPDH: Data on the interaction with the enzyme and the impact on its intracellular content.

This data is related to our paper "Small molecules preventing GAPDH aggregation are therapeutically applicable in cell and rat models of oxidative stress" (Lazarev et al. [1]) where we explore therapeutic properties of small molecules preventing GAPDH aggregation in cell and rat models of oxidative stress. The present article demonstrates a few of additional properties of the chemicals shown to block GAPDH aggregation such as calculated site for targeting the enzyme, effects on GAPDH glycolytic activity, influence on GAPDH intracellular level and anti-aggregate activity of pure polyglutamine exemplifying a denatured protein.

Small molecules preventing GAPDH aggregation are therapeutically applicable in cell and rat models of oxidative stress.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most abundant targets of the oxidative stress. Oxidation of the enzyme causes its inactivation and the formation of intermolecular disulfide bonds, and leads to the accumulation of GAPDH aggregates and ultimately to cell death. The aim of this work was to reveal the ability of chemicals to break the described above pathologic linkage by inhibiting GAPDH aggregation. Using the model of oxidative stress based on SK-N-SH human neuroblastoma cells treated with hydrogen peroxide, we found that lentivirus-mediated down- or up-regulation of GAPDH content caused inhibition or enhancement of the protein aggregation and respectively reduced or increased the level of cell death. To reveal substances that are able to inhibit GAPDH aggregation, we developed a special assay based on dot ultrafiltration using the collection of small molecules of plant origin. In the first round of screening, five compounds were found to possess anti-aggregation activity as established by ultrafiltration and dynamic light scattering; some of the substances efficiently inhibited GAPDH aggregation in nanomolar concentrations. The ability of the compounds to bind GAPDH molecules was proved by the drug affinity responsive target stability assay, molecular docking and differential scanning calorimetry. Results of experiments with SK-N-SH human neuroblastoma treated with hydrogen peroxide show that two substances, RX409 and RX426, lowered the degree of GAPDH aggregation and reduced cell death by 30%. Oxidative injury was emulated in vivo by injecting of malonic acid into the rat brain, and we showed that the treatment with RX409 or RX426 inhibited GAPDH-mediated aggregation in the brain, reduced areas of the injury as proved by magnetic resonance imaging, and augmented the behavioral status of the rats as established by the "beam walking" test. In conclusion, the data show that two GAPDH binders could be therapeutically relevant in the treatment of injuries stemming from hard oxidative stress.