E. M. Lazareva

Lignin - a useful bioresource for the production of sorption-active materials.

Sorption characteristics of acid hydrolysis lignins, commercial by-products of wood conversion to fuel ethanol, and their nitrogen-containing derivatives have been examined to determine the most suitable areas of application of lignin-based sorbents. The results obtained have shown that the sorption capacity for organic contaminants of an aromatic nature increases significantly as a result of the modification of hydrolysis lignins with quaternary ammonium compounds. The amination of lignin with epoxy amines enhanced its sorption activity towards heavy metals. Aminolignins have a high sorption capacity for bile acids and cholesterol. Sorption properties of nitrogen-containing lignin derivatives are such that they can be proposed for use in the environment-protection field and as enterosorbents.

Uptake and accumulation of multiwalled carbon nanotubes change the morphometric and biochemical characteristics of Onobrychis arenaria seedlings

We have studied the effect of the engineered nanomaterial Taunit, containing multiwalled carbon nanotubes (MWCNTs), on the growth of Onobrychis arenaria seedlings and investigated whether affected plants uptake and accumulate MWCNTs. We found that 100 μg/mL and 1000 μg/mL of Taunit stimulated the growth of roots and stems, and enhanced the peroxidase activity in these parts of plants. Microscopy studies showed the presence of MWCNTs in the root and leaf tissues of seedlings exposed to Taunit, suggesting that MWCNTs have a capacity to penetrate the cell walls, accumulate in roots and translocate to the leaves. Thus the stimulating effect of MWCNTs on seedlings of O. arenaria may be associated with the primary uptake and accumulation of MWCNTs by plant roots followed by translocation to the other plant tissues.

Time and cell cycle dependent formation of heterogeneous tubulin arrays induced by colchicine in Triticum aestivum root meristem

We have investigated the appearance and reorganization of tubulin‐containing arrays induced by colchicine in the root meristem of wheat Triticum aestivum, using immunostaining and electron microscopy. Colchicine caused depolymerization of microtubules and formation of tubulin cortical strands composed of filamentous material only in C‐mitotic cells. After prolonged exposure to the drug, both interphase and C‐mitotic cells acquired needle‐type bundles, arranged as different crystalloids and/or macrotubules. The unmodified tyrosinated form of α‐tubulin was detected within microtubules in control cells, but was not found within cortical strands. It was identified, however, within needle‐type bundles. The modified acetylated form of α‐tubulin, which was absent in control cells, was detected within needle‐type bundles. Thus, cortical strands were transitory arrays, transformed into needle‐type bundles during prolonged exposure to colchicine. Cortical strands appeared in a cell cycle‐dependent manner, whereas needle‐type bundles were cell cycle stable arrays. The diverse morphological organization, intracellular distribution and stability of tubulin‐containing arrays may be associated with heterogeneity of α‐tubulin isoforms. We assume that non‐microtubular arrays substitute for microtubules in conditions where normal tubulin polymerization is inhibited.

A high molecular weight polypeptide cross-reacting with the antibodies to the dynein heavy chain localizes to the subset of Golgi complex in higher plant cells.

Antibodies were produced against fragments of the microtubule‐binding domain and the motor domain of the dynein heavy chain from Dictyostelium discoideum to probe whole cell extracts of root meristem cells of wheat Triticum aestivum. In plant extracts, these antibodies cross‐reacted with a polypeptide of high molecular weight (>500 kDa). The antibodies bound to protein A‐Sepharose precipitated high molecular weight polypeptide from cell extracts. Immunofluorescence showed that the antibodies identified various aggregates inside cells, localized at the perinuclear area during interphase to early prophase, at the spindle periphery and polar area during mitosis, and in the interzonal region during phragmoplast development. Some aggregates were also co‐labeled by markers for the Golgi apparatus. Thus, we found in higher plant cells a high molecular weight antigen cross‐reacting with the antibodies to motor and microtubule‐binding domains of dynein heavy chains. This antigen is associated with aggregates distributed in the cytoplasm in cell cycle‐dependent manner. A subset of these aggregates belongs to the Golgi complex.

Trifluralin-induced disorganization of microtubular cytoskeleton alters the development of roots in Hordeum vulgare L.

The extensive use of herbicides in agriculture becomes an important factor in environmental pollution, especially in case of slowly degradable compounds. Some agents act on plants during a long period of time, even if a very low concentration of the herbicide remains in the soil. Here, we investigated the toxicological effect of a low concentration of dinitroaniline herbicide, trifluralin, on growing seedlings of Hordeum vulgare L. Trifluralin in concentration of 1 microg/ml inhibited root growth. The mitotic activity of meristematic cells was suppressed due to the retardation of metaphase progression--alteration that can be caused by cytoskeleton disorder. Using antibodies to alpha-tubulin, we investigated the distribution of microtubules in root meristem cells. During all stages of mitosis, the highly regular system of microtubular cytoskeleton observed in control cells was slightly disorganized. An examination of root structure using light and electron microscopy demonstrated that the cell walls did not form normally during cell division that led to the appearance of large multinucleated cells. Also, the premature (pathological) cell differentiation was induced by trifluralin. A part of differentiating cells showed intracellular structural changes that are consistent with programmed cell death. It seems that the development of alterations in trifluralin-treated roots was due to the microtubular cytoskeleton disorganization.

Antipodal complex development in the embryo sac of wheat

Dynamics of an antipodal complex formation in wheat (Tritiñum aestivum L.) has been observed in detail using a reconstruction of serial semifine sections. Three consecutive crucial stages have been identified in the development of the antipodal complex: (1) proliferation of initial cells, (2) growth and functional differentiation of antipodal cells, and (3) cell apoptosis. Specific features of the mitotic division of antipodal cells have been characterized. It has been shown that the structure of interphase nuclei and mitotic chromosomes of proliferating antipodal cells is similar to that of nucellar cells surrounding the embryo sac. According to the reconstruction of appropriately oriented serial sections, the division of antipodal cells is asynchronous. DNA content in differentiated antipodal cells has been determined by a cytophotometric analysis; in the case of a mature embryo sac, the ploidy of antipodal cells varied from 8 to 32C. Proliferation and DNA endoreduplication processes in the antipodal complex proceed at different time; the second process starts only after the termination of the first one. DNA endoreduplication is accompanied by total chromatin remodeling; as a result, giant chromosomes are formed in the nuclei of antipodal cells. The final stage of the antipodal complex development is programmed cell death or apoptosis. A model for the structural organization of an antipodal complex has been proposed based on the layer arrangement of cells. The secretory activity of antipodal cells directed towards the endosperm syncytium has been detected for the first time. The analysis of “truncated” ovules with an undeveloped endosperm has shown that developing endosperm can be a possible inductor, which stimulates the functional activity of antipodal cells and triggers their terminal differentiation. The obtained results evidence the functional role of antipodal cells in the development of the endosperm and embryo.

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L.

We have studied the response of interphase and mitotic microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) to cold stress (1 h at 0°C) and acclimation to cold (3–48 h at 0°C). We show that, in general, interphase microtubules are more resistant to cold then mitotic arrays in both cultivars. During cold stress, no changes are detected in the microtubule system of interphase cells of spring wheat, whereas the density of endoplasmic microtubules increases in interphase cells of winter wheat. During mitosis, the density of the kinetochore fibers of the spindle decreases in the cells of both cultivars, but it is prevailing in the cells of spring cultivar of wheat. During acclimation to cold, the disorganization of the cortical microtubule bundles and the enhanced growth of the endoplasmic microtubule network, which is comprised of microtubule converging centers, are observed in cells of both cultivars. However, the mitotic microtubule systems of winter and spring cultivars respond differently to cold acclimation. During prophase, a diffuse tubulin “halo,”followed by the assembly of microtubule converging centers, accumulate at the perinuclear area in the cells of winter wheat. In cells of spring cultivar, the prophase spindle is only detected during initial stages of cold acclimation. During metaphase, aberrant mitotic spindles, abnormal metaphase plates, and the excessive appearance of microtubule converging centers are observed in cells of both cultivars. Acclimation induces the disorganization of the phragmoplast and the formation of multiple microtubule converging centers during telophase in the cells of both cultivars. Microtubule converging centers are detected at the perinuclear area of daughter cells in winter wheat and in the cortical cytoplasm in spring wheat. The excessive formation of microtubule converging centers suggests the activation of microtubule assembly during prolonged exposure to low temperature. Our data also demonstrates common pathways of microtubule response to cold treatment (0°C).

High molecular weight protein detected in higher plant cells by antibodies against dynein is associated with vesicular organelles including Golgi apparatus

The cytoplasmic dynein is a multisubunit complex driving organelles along microtubules to their minus-end. We used antibodies against two functional domains (motor and microtubule-binding) of one of principal components of the complex—dynein heavy chain of slime mould Dictyostelium discoideum—to test root meristem cells of wheat Triticum aestivum. The antibodies reacted with a high molecular weight protein (>500 kDa) in the total cell extract and the band recognized by the antibodies in plant extracts had a lower electrophoretic mobility than the high molecular weight band of mammalian dynein. Antibodies coupled to protein A-Sepharose precipitated the high molecular weight protein from the purified cell extracts. Immunocytochemical analysis demonstrated that the antigen recognized by antibodies against dynein heavy chains is associated with the vesicles whose localization depends on the cell cycle stage. The antigen-positive vesicles were localized to the perinuclear region in interphase and early prophase, to the spindle periphery and to spindle pole region during mitosis, and to the interzonal region in the period of fragmoplast and cell plate formation. Some antigen-positive vesicles also reacted with antibodies against Golgi protein markers. The obtained data indicate that higher plant cells contain a high molecular weight protein interacting with antibodies against the motor and microtubules-binding domains of Dictyostelium dynein heavy chain. The revealed antigen was associated with the vesicular structures in the cytoplasm including the Golgi apparatus.

Synergistic effects of mycelial fungus (Pleurotes ostreatus) extracts and some cytostatic drugs on proliferation and apoptosis in transformed human cells

Effects of mycelial fungus (Pleurotes ostreatus) extracts used separately and in combination with the cytostatic drugs doxorubicine and cyclophosphamide on cultured transformed human cells (HeLa and myeloid leukemic cells) were investigated. Cell viability was assessed by calculating mitotic and apoptotic indexes and other characteristics of dividing cells. The functional status of cell membranes was estimated by staining with Trypan blue. Individual application of mycelium extracts failed to induce apoptosis, but caused abnormal segregation of chromosomes in metaphase cells and formation of chromosome bridges in anaphase and telophase. Combined application of P. ostreatus extracts and cyclophosphamide produced pronounced synergistic effect, which depended on reagent concentration and treatment protocol and was manifested in partial suppression/enchancement of cell proliferation and in drastic increase of the apototic index. The data obtained are discussed within the framework of a hypothesis on intracellular targets for P. ostreatus extracts.

Reorganization of interphase microtubules in root cells of Medicago sativa L. during acclimation to osmotic and salt stress

We examined the organization of microtubule system of interphase cells in roots of Medicago sativa L. during acclimation to salt and osmotic stress at different concentrations of NaCl, Na2SO4, and mannitol. We identified morphological changes of tubulin cytoskeleton in different root tissues during the acclimation to salt and osmotic stress: (1) decreased density of the cortical microtubule network, (2) random orientation of cortical microtubule bundles, (4) thickening of the bundles, (3) nonuniform density of the bundles, (4) fragmentation of the bundles, and (5) formation of microtubule converging centers. Network thinning and thickening of the bundles were observed both under osmotic and salt stress. Random orientation of cortical microtubules was visualized under osmotic stress but not during salt stress. Fragmentation of microtubule bundles took place under salt stress with a high concentration of mannitol. Formation of microtubule converging centers was common under prolonged action of sodium sulfate, less evident under sodium chloride, and not found after mannitol treatment. Our data show that, in alfalfa root cells, cortical microtubules rearrange not only in response to different ions, but also to osmotic pressure. Thus, the signaling pathways and molecular mechanisms inducing reorganization of the microtubule system may be triggered by sodium cations, as well as by sulfate and chloride anions at concentrations that do not cause irreversible cell damage.