Developmental Responses to Drought and Abscisic Acid in Sunflower Roots: 2. MITOTIC ACTIVITY

Mitotic activity was studied in the root apices of aeroponicallygrown sunflower seedlings (Helianthus annum L. var. RussianGiant) which were draughted or treated with abscisic acid (ABA)over a 7 d period. Labelling index (LI) and mitotic index (MI)were scored from autoradiographs of median longitudinal sectionsof [³H] methyl-thymidine treated root apices. Both drought stressand ABA-treatment (at a concentration of 10^–2 mol m^–3inhibited DNA synthesis and mitosis within the first 6 h oftreatment. The depression of mitotic activity was first evidentin the proximal regions of the meristem (1000–1500 µmfrom the cap junction). This was followed by a general depressionof mitotic activity throughout the meristem which was, in turn,followed by a partial recovery of mitotic activity in the distalregions of the meristem. The beginning of this partial recoverywas concurrent with the activation of the quiescent centre (QC).Treatment with lower concentrations of ABA (10^–3 mol m^–3and 10^–4 mol m^–3) also inhibited mitotic activity.Exogenous supplements of sucrose to the plant did not alleviatethe inhibition of mitotic activity by drought or ABA. Thesefindings support the hypothesis that ABA mediates drought-inducedchanges in the primary development of sunflower roots. Key words: Abscisic acid, drought, mitotic activity     

Restoration of cell growth and proliferation in the wheat roots after their inhibition by nickel sulfate

The dynamics of cell growth and proliferation restoration in different tissues and quiescent center (QC) in the wheat (Triticum aestivum L.) seedling roots and also the differentiation of rhizodermal cells and lateral root initiation after 48-h treatment with 100 μM NiSO₄ were studied. Within 24 h after nickel removal from medium, root growth was resumed due to the increase in the rate of cell growth in the meristem and the region where cell elongation started in control roots. Stimulation of cell proliferation was restored in the main part of the meristem and later in the initial cells of the files and QC. Cell proliferation was not observed in the QC. The time of cell proliferation resumption in the roots and in tested tissues depended on the degree of their injury by nickel treatment. In most tested roots, DNA synthesis and cell division were restored in 32 h. In the cells leaving the meristem due to the resumption of their growth and proliferation, growth of root hairs started. In 48 h, the number of roots with perished cells in the rhizodermis in the meristem was sharply increased and the regeneration of the damaged region by the cells of outer cortex was observed. Only after the appearance of root hairs, the cells coming from the meristem started to elongate. In most roots, the formation of the new elongation zone occurred in 56 h. During its formation, the initiation of lateral root primordia was shifted in the basipetal direction. It was concluded that the cessation of cell growth and proliferation under the influence of high concentration of heavy metal (HM) ions is not lethal for the root. At the action of toxic HM concentrations, the plant strategy is the maintenance of meristematic cell capacity for cell growth and proliferation resumption. The cellular mechanism of this capacity maintenance is the transition of meristematic cells from G₁ phase to dormancy due to growth inhibition and the inhibition of the transition to DNA synthesis.     

Morphological changes induced in fungi by antibiotics

In tests of 31 antibiotics, 29 inhibited growth ofBotrytis cinerea and of these, 18 induced morphological changes. Terminal and lateral branching of the hyphae was induced by actinomycin D, aspergillic acid, citrinin, cyanein, cycloheximide, desertomycin and polyene antibiotics. Curling of the hyphae was induced by griseofulvin and narrowing of the hyphae by citrinin. Some antibiotics at different concentrations produced several types of morphological changes. For example, aspergillic acid, desertomycin and flavofungin also induced terminal bulging of the hyphae. Growth of the dimorphic fungusPaecilomyces viridis was inhibited by 24 antibioties, nine of which induced morphological changes. Branching of the hyphae was induced by azalomycin F, citrinin, eyanein, desertomycin, patulin, rugulosin and trichothecin. Griseofulvin had a curling effect. Except for rugulosin, the above antibiotics, in higher concentrations, induced yeast-like growth ofPaecilomyces viridis. Morphological changes were also induced by inhibitors of RNA and protein synthesis and by antibiotics injuring the cell membranes. Antibiotics with different mechanisms of action induced similar morphological changes.     

The effect of butyrate on the cell cycle in root meristems of Pisum sativum

Sodium butyrate at 5 mM in aerated White's medium reduced the mitotic index in root meristems of seedlings of Pisum sativum to < 1% after 12 h. This effect was lessened as the butyrate concentrations were lowered. The fraction of the root meristem nuclei in G2 increased to ~ 70% after 12 h in butyrate. After 12 h exposure to butyrate, seedlings transferred lo medium without butyrate gradually re-established their normal root meristem mitotic pattern, with a burst of mitosis at 10 h after the transfer. Even a brief exposure to butyrate inhibited DNA synthesis, and nuclei released from butyrate exposure were still unable to resume normal DNA synthesis even after 12 h. This information suggests that butyrate halts progression through the cell cycle by arresting meristem nuclei in G2 and inhibiting DNA synthesis.     

Short-chain fatty-acid-induced effects on the cell cycle in root meristems of Pisum sativum

Propionic acid and valeric acid at 1 m M reduced the mitotic index of root meristem cells of Pisum sativum to <1% after 12 h in aerated White's medium. After 12 h exposure to either acid, seedlings transferred to fresh medium resumed their normal mitotic index 12 h after transfer, with a burst of mitosis at 8 h. Exposure times of 8 h to either acid inhibited DNA synthesis, and nuclei released from either propionic or valeric acid inhibition were still unable to resume normal DNA synthesis after 12 h. Neither acid significantly altered the distribution of meristematic cells in G1 and G2 after 12 h. Propionic acid at 1 m M reduced the uptake of [¹⁴C]-leucin but conversion rates to protein were constant regardless of whether any acid was present. Another longer fatty acid, caprylic acid, at 1 m M did not significantly reduce the mitotic index nor did 1 m M benzoic acid, another organic acid. This information suggests that only the short-chain fatty acids, propionic acid and valeric acid, limit progression through the cell cycle by inhibiting DNA synthesis and arresting cells in G1 and G2 in a manner similar to butyric acid, a known cell arresting agent.     

Cell Division Heterogeneity in the Primary Meristems of Pisum sativum Root Tips after Excision

Root tips (1.0 cm) were excised, transferred to culture medium,sampled at intervals, embedded in paraffin and sectioned. Themitotic index in each primary meristem in the 2.0 mm tips wasreduced immediately after excision. After 8–12 h mitosisresumed. The position of mitotic figures in the protoderm, groundmeristem, procambium, and root cap was determined. The greatestnumber of mitotic figures occurred in the procambium, and uponexcision the smallest percent reduction also occurred in thisregion. The ground meristem showed the greatest reduction inmitotic figures. After excision, the relative contribution tothe total number of mitotic figures increased in the procambiumtissue and decreased in the ground meristem. Cell division inall primary meristems of the root tip is reduced after tip excision,but the ground meristem (cortical region) is the primary targettissue.     

Structural architectonics of apical root meristems in coherence with the quantitative assessment of its damage by radiation

The dose dependencies of the aberrant anaphases frequency in the root meristem in 48 hours after irradiation in the range of doses of 4-10 Gy is characterized by threshold and plateau at 33% aberrant anaphase. The plateau indicates the activation of the recovery processes. Topology of cell rows in the primary meristem of the dose to 8 Gy are conserved and recovered damages. New cell rows are formed by local cell pools in the distal meristem, pericycle cells and subepidermy. It grows by intrusive character displacing the rows of damaged cells. Apparently the competition between clones of normal and aberrant cells plays the primary role in the mechanisms of recovery. Resulting to competition the promotion of aberrant cells to the extension zone is slowed down or blocked. So critical level of damage of the root apical meristem was defined about 50% of aberrant anaphase. Exceeding of this level leads to lethal consequence for meristem and it is accompanied by the inclusion of more radical process of restoration through regeneration. Regeneration leads to complete replacement of the apex tissues including the extension zone.     

Effects of antibiotics on the life cycle ofNeurospora crassa

Some antibiotics and synthetic inhibitors affect, in several ways, the life cycle ofNeurospora crassa (germination of conidia → myceliar growth → formation of conidia). Bikaverin, cyanein, scopathrioin and phenethyl alcohol retard the germination of conidia, without inhibiting it completely. 5-Fluorouracil, ramihyphin A and zygosporin A (cytoohalasin D) do not inhibit the germination. Bikaverin brings about a thickening of the hyphae of growing mycelium. Ramihyphin A, cyanein, scopathricin and zygosporin A stimulate the ramification of hyphae while 5-fluorouracil and phenethyl alcohol do not affect the myceliar morphology apart from their inhibitory effect on growth. Actinomycin D, 5-fluorouracil, cycloheximide, ramihyphin A and partially also sodium iodoacetate inhibit to a different degree the photoinduced formation of conidia. The inhibition by 5-fluorouracil is very conspicuous when the agent is present during the photoinduction but considerably weaker when it is applied 2 h after the photoinduction.     

Morphologic and phenotypic changes of human neuroblastoma cells in culture induced by cytosine arabinoside

The effects of cytosine-arabinoside (ARA-C) on the growth and phenotypic expression of a new human neuroblastoma (NB) cell line (GI-ME-N) have been extensively tested. Low doses of ARA-C allowing more than 90% cell viability induce morphological differentiation and growth inhibition. Differentiated cells were larger and flattened with elongated dendritic processes; such cells appeared within 48 h after a dose of ARA-C as low as 0.1 micrograms/ml (about 1000-fold lower than the conventional clinic dose). The new morphological aspect reached the maximum expression after 5-6 days of culture being independent from the addition of extra drug to the culture. A decrease in [3H]thymidine incorporation was also observed within 24 h and the cell growth was completely inhibited on the sixth day. Moreover, ARA-C strongly inhibited anchorage-independent growth in soft agar assay. Membrane immunofluorescence showed several dramatic changes in NB-specific antigen expression after 5 days of treatment with ARA-C. At the same time ARA-C also modulated cytoskeletal proteins and slightly increased catecholamine expression. These findings suggest that noncytotoxic doses of ARA-C do promote the differentiation of GI-ME-N neuroblastoma cells associated with reduced expression of the malignant phenotype.     

Griseofulvin: Association with tubulin and inhibition of in vitro microtubule assembly

Griseofulvin—shown previously to disrupt the mitotic apparatus $\text{in vivo}$—inhibited the $\text{in vitro}$ microtubule assembly reaction completely at 8 × 10^?4M griseofulvin. In a gel filtration assay, randomly tritiated griseofulvin associated stoichiometrically with purified tubulin, as determined by chromatography on Sephadex G-25. No detectable drug binding was observed when bovine serum albumin was used as a control in an identical column assay. Both gel filtration chromatography and a kinetic analysis of the inhibition of assembly by griseofulvin suggest that the drug interacts directly and stoichimetrically with the tubulin dimer, and that the interaction is both rapid and independent of temperature.     

Effect of tungstate on pea root growth and protein tyrosine phosphorylation

Tungsten belong to heavy metal group, which physiological, biochemical, and molecular action mechanisms are essentially unstudied despite metal wide application in light, heavy, and military industries and the gradual accumulation in the environment. Protein phosphorylation/dephosphorylation (one of the most important posttranslational modifications) is a highly conserved mechanism of intracellular signaling and regulation of many processes of cell activity. Protein tyrosine phosphorylation/dephosphorylation is required for the cell cycle processing, plant growth and differentiation. In this work, the effects of sodium tungstate on pea (Pisum sativum L. cv. Truzhenik) root growth, protein tyrosine phosphorylation, and phosphatase activity in the roots were studied. It was shown that sodium tungstate suppressed growth, changed the mitotic index in the root meristem, and delayed cells at some mitosis phases. Under the influence of tungstate, hydrogen peroxide accumulated in the roots and phosphatase activity was inhibited. It was established by two-dimension electrophoresis and immunoblotting with the highly specific to phosphotyrosine antibody (PY20) that tungstate treatment increased both the number of such proteins and their specific phosphorylation. It is supposed that the inhibition of protein tyrosine phosphatases was one of the reasons for tungstateinduced pea root growth inhibition.     

Decrease in Ca2+-Activated K+ Conductance in Differentiated C6-Glioma Cells

Ca2+-activated K+ channels were studied in C6-glioma cells in an attempt to correlate changes in expression with cell proliferation and differentiation. In this study, we treated C6-glioma cells with thapsigargin for 48 h. Cell proliferation was markedly inhibited, and cell morphology changed from round to a spindle differentiated shape. Furthermore, intracellular calcium concentration was initially increased during acute treatment with thapsigargin. The internal [Ca2+]i pool was eventually depleted after a 48-h thapsigargin treatment. We have characterized Ca2+-activated K+ currents in less differentiated C6 cells. After differentiation of C6 cells induced by thapsigargin, Ca2+-activated K+ currents were selectively suppressed. These data lend further support to the notion that the expression of Ca2+-activated K+ channels is intimately associated with the proliferation of C6-glioma cells, and the suppression of Ca2+-activated K+ channels coincides with the inhibition of proliferation and subsequent induction of cell differentiation.     

The inhibitory effect of some lectins on the differentiation of Friend erythroleukemia cells

Growth and differentiation of Friend cells can be inhibited by treatment with concanavalin A and wheat-germ agglutinin. This inhibition is specific for cells that are in the early stages of the differentiation process (24–48 h after the addition of dimethyl sulfoxide) and is reversible upon treatment with the sugars competitive for these lectins. These results suggest a regulatory role for some plasma membrane-bound glycoproteins early in the differentiation of Friend erythroleukemia cells induced by dimethyl sulfoxide.     

Short term cultivation of isolated barley roots and their mitotic activity

Roots were excised from barley embryos cultivated in the complete liquid nutrient solution and cultivated in the same nutrient solution separately. The excised roots continued their growth but a progressive decrease in the growth rate was observed. There was a considerable short-term drop of the mitotic activity immediately after excision, which was followed by a compensatory increase and then equilibrium was reached 12 h after excision. During the next at least three days, the mitotic index of isolated barley roots varied between 5–6.5%, which is slightly lower than the mitotic index of the root meristems of isolated barley embryos under identical conditions. The mitotic cycle index of isolated barley roots and the size of the root meristem later decreased gradually.     

Regulation by calcium of the proliferation of heart cells from young adult rats

Summary Cells of primary and secondary cultures of rat heart ventricle were grown in a medium supplemented with homologous plasma to avoid the non-specific proliferogenic effects of foreign sera. Specific chelation of calcium from the medium arrested the cells' progression through their cycle at the G1 phase. A permanent or brief restoration of the extracellular calcium level (48 hr later) was followed, after a 5 to 6 hr delay, by a burst of DNA synthesis, and the resumption of mitotic activity. NRCC No. 14976.     

Effects of γ-interferon on the growth, morphology, and membrane and cytoskeletal proteins expression of LAN-1 cells

The effects of gamma-interferon (gamma-IFN) on the growth, morphology, and phenotypic expression of the human neuroblastoma (NB) cell line, LAN-1, have been extensively tested. Low doses of gamma-IFN allowing more than 90% cell viability induce morphological differentiation and growth inhibition. Cells exposed to gamma-IFN significantly decreased their growth rate, became smaller and poligonal, and sprouted long cellular processes with varicosities along their course, typical of the neurites seen in differentiated NB cells; morphological changes appeared within 48 h of culture with 1,000 U/ml gamma-IFN. The new morphological aspect reached the maximum expression after 6 days of culture, becoming more evident when fresh drug was added after 2 days of culture. A decrease in [3H]thymidine incorporation was also observed within 24 h; cell growth was completely inhibited at the 6th day. Membrane immunofluorescence showed several changes in NB-specific antigen expression after 6 days of treatment with gamma-IFN. At the same time gamma-IFN also modulated cytoskeletal proteins. These findings suggest that noncytotoxic doses of gamma-IFN do promote the differentiation of LAN-1 neuroblastoma cells which is associated with the reduced expression of the malignant phenotype.     

Regulation of differentiation of the dimorphic fungusPaecilomyces viridis by nitrogen sources,antibiotics and metabolic inhibitors

The effect of nitrogen and carbon sources, vitamins, antibiotics and metabolic inhibitors on growth and differentiation ofPaecilomyces viridis was investigated. Sodium nitrate,l-asparagine,l-proline and peptone were found to be suitable nitrogen sources for mycelial growth (M) in a synthetic medium with glucose.Paecilomyces viridis could also grow slowly in a synthetic medium containing benzylpenicillin or bacitracin as the only nitrogen sources and very slowly even in a medium with polymyxin as the nitrogen source. Ammonium salts, area,l-arginine,d, l-aspartic acid andl,-serine were found to support intensive sporulation. Partially yeast-like growth (Y) was facilitated by NaNO₂, (NH₄)₂SO₄, NH₄NO₃, urea,d, l-alanine,l-arginine,d, l-aspartic acid,l-cysteine,l-glutamic acid andl-serine. Partially yeastlike growth could be observed in a medium with peptone and at an initial pH of 2. The following compounds appear as suitable carbon sources for mycelial growth:d-glucose,d-galactose,d-mannose, maltose, sucrose, chitin andd-mannitol. No changes in morphology could be detected on any of the 25 used carbon sources in a synthetic medium with NaNO₃. Yeast-like growth was induced by the antibiotics azalomycin F, cyanein (brefeldin A), griseofulvin and monorden (radicicol). After removal of the antibiotics, mycelial growth was restored. Sporulation was stimulated by chloramphenicol, 2-deoxy-d-glucose, furancarboxylic acid and stipitatic acid. Deformation of phialides was observed after treatment with actinomycin D, amphotericin B, boromycin, citrinin, cycloheximide, cytochalasin D, fungicidin and scopathricin. Microcyclic conidiation or growth of phialides directly from conidia were induced by cycloheximide, desertomycin, ethidium bromide and 5-fluorouracil.     

斑蝥素对粘虫几种代谢酶及多酚氧化酶的影响

为进一步探讨斑蝥素的杀虫作用机理, 本研究采用叶碟饲喂法处理粘虫Mythimna separata（Walker）5龄幼虫, 测定了饲喂处理后6, 12, 24, 36和48 h试虫体内羧酸酯酶(CarE)、酸性磷酸酯酶(ACP)、碱性磷酸酯酶(ALP)、谷胱甘肽 S-转移酶(GST)和细胞色素P450 Ο-脱甲基酶及多酚氧化酶(PPO)的活性。结果表明: 斑蝥素可显著激活羧酸酯酶, 处理后48 h酶比活力最大, 为同期对照的1.68倍; 酸性磷酸酯酶在处理后6和12 h活性变化不明显, 处理24 h后逐渐被激活, 且与对照差异显著(P＜0.05); 明显抑制碱性磷酸酯酶, 且随着处理时间的延长, 抑制作用增强; 对Ο-脱甲基酶表现为先抑制后激活的趋势; 谷胱甘肽S-转移酶表现出先激活后抑制的影响; 离体活体条件下均可显著抑制PPO活性。可见, 斑蝥素可明显影响昆虫的代谢酶系, 且其对碱性磷酸酶和多酚氧化酶的抑制作用可能与其毒杀作用有关。     

Osteogenin inhibits proliferation and stimulates differentiation in mouse osteoblast-like cells (MC3T3-E1)

Osteogenin, a novel bone differentiation factor, was recently purified and characterized. We examined its effect on the proliferation and differentiation of MC3T3-E1 osteoblast-like cells. Cell proliferation was inhibited the first 48 h after addition of osteogenin, and this effect was independent of serum. Osteogenin did not influence the cell morphology. Alkaline phosphatase promptly increased in a dose and time-dependent manner and appeared to be specific. Treatment with TGF-beta 1 resulted in inhibition of alkaline phosphatase activity, and was reversed by osteogenin within 48 h. Cell cultures treated with osteogenin for 72 h after confluence became responsive to parathyroid hormone. Synthesis of collagenous proteins was stimulated by osteogenin. The present results demonstrate a significant influence of osteogenin on the differentiation of osteogenic phenotype in MC3T3-E1 cells in vitro.     

Cell cycle regulation in the course of nodule organogenesis in Medicago

The molecular mechanisms of de novo meristem formation, cell differentiation and the integration of the cell cycle machinery into appropriate stages of the developmental programmes are still largely unknown in plants. Legume root nodules, which house nitrogen-fixing rhizobia, are unique plant organs and their development may serve as a model for organogenetic processes in plants. Nodules form and are essential for the plant only under limitation of combined nitrogen in the soil. Moreover, their development is triggered by external mitogenic signals produced by their symbiotic partners, the rhizobia. These signals, the lipochitooligosaccharide Nod factors, act as host-specific morphogens and induce the re-entry of root cortical cells into mitotic cycles. Maintenance of cell division activity leads to the formation of a persistent nodule meristem from which cells exit continuously and enter the nodule differentiation programme, involving multiple cycles of endoreduplication and enlargement of nuclear and cell volumes. While the small diploid 2C cells remain uninfected, the large polyploid cells can be invaded and, after completing the differentiation programme, host the nitrogen-fixing bacteroids. This review summarizes the present knowledge on cell cycle reactivation and meristem formation in response to Nod factors and reports on a novel plant cell cycle regulator that can switch mitotic cycles to differentiation programmes.