Metabolism of diclofop-methyl (methyl-2-[4-(2',4'-dichlorophenoxy)phenoxy] propanoate) in cell suspensions of diploid wheat (Triticum monococcum)

The metabolism of the herbicide, diclofop-methyl (methyl-2-[4-(2′,4′-dichlorophenoxy)phenoxy]propanoate, in cell suspensions of resistant diploid wheat (Triticum monococcum L.) was determined 1, 8, and 24 h after treatment with ¹⁴C-diclofop-methyl. The ¹⁴C-labeled products were identified by thin layer chromatographic comparisons to appropriate standards. Eight hours after treatment with 5 μM diclofop-methyl in 0.8% acetone (neither of which were toxic to the cell suspensions) 87.2% (84.0% methanol soluble, 3.2% methanol insoluble) of the total ¹⁴C recovered (90.4%) was in the cells and 12.8% was in the medium. Major metabolites found in methanol extracts of the cells were diclofop (2-[4-(2′,4′-dichlorophenoxy)phenoxylpropionic acid), diclofop hydroxylated at an undetermined position on the 2,4-dichlorophenyl ring (ring-OH diclofop), and conjugates of ring-OH diclofop. Acid hydrolysis of the conjugated metabolite(s) yielded ring-OH diclofop and diclofop. Twenty-four hours after treatment 70–75% of the total ¹⁴C recovered was present as conjugated metabolites. With the exception of ring-OH diclofop, all metabolites present in the cells were also recovered from the medium. A metabolite found in low concentrations in the medium that yielded diclofop upon hydrolysis was identified as an ester conjugate. Toxic concentrations of diclofop-methyl (10 and 20μM) had no effect on the metabolism of the herbicide, although the rate of uptake was slower than for cells treated with 5 μM herbicide. The products of diclofop-methyl metabolism in cell suspensions of T. monococcum were compared to previous data from T. aestivum intact plant metabolism of diclofop-methyl.     

Growth of mouse fibroblasts in the presence of irradiated and lyophilized monolayers of the same type of cells

Certain cells, such as 3T3 mouse embryo fibroblasts, are inhibited from dividing when they grow to a characteristic cell density on a surface in tissue culture. We asked whether the inhibition of cell division could be attributed to the inert chemical composition of neighboring cells, that is, whether the residues of lyophilized cells retained the ability to inhibit the division of normal cells. In addition, we wanted to know whether cells in which DNA synthesis was imparied by irradiation would retain the capacity to effectively inhibit normal cells. To answer these questions, confluent and non-confluent layers of 3T3 cells were prepared in tissue culture dishes and the cells were either lyophilized or irrariated in situ. Fresh 3T3 cells were then added to these prepared layers and their growth was followed using radioactive label. There was no growth of added cells on the confluent monolayers of either untreated or irradiated cells. Growth was unimpeded on the monolayers of lyophilized cells. When cells were added to non-confluent cultures of either normal or irradiated cells the added cells grew until they had covered the remaining surface of the culture dish and had come into contact with the pre-existing cells. In the discussion, consideration is given to the role of available surface over which the cells can spread as well as to the possible interactions between neighboring cells.     

Bialaphos selection of stable transformants from maize cell culture

Summary Stable transformed Black Mexican Sweet (BMS) maize callus was recovered from suspension culture cells bombarded with plasmid DNA that conferred resistance to the herbicide bialaphos. Suspension culture cells were bombarded with a mixture of two plasmids. One plasmid contained a selectable marker gene, bar, which encoded phosphinothricin acetyl transferase (PAT), and the other plasmid encoded a screenable marker for -glucuronidase (GUS). Bombarded cells were selected on medium containing the herbicide bialaphos, which is cleaved in plant cells to yield phosphinothricin (PPT), an inhibitor of glutamine synthetase. The bialaphos-resistant callus contained the bar gene and expressed PAT as assayed by PPT inactivation. Transformants that expressed high levels of PAT grew more rapidly on increasing concentrations of bialaphos than transformants expressing low levels of PAT. Fifty percent of the bialaphos-resistant transformants tested (8 of 16) expressed the nonselected gene encoding GUS.     

Cholera toxin stimulates division of 3T3 cells.

Cholera toxin was used in an attempt to inhibit epidermal growth factor stimulated 3T3 cell division. Instead, cholera toxin alone at low concentrations (10(-10) M), was able to stimulate cell division and could augment EGF stimulated cell division. The mitogenic effect of cholera toxin can occur despite a dramatic increase in the intracellular levels of cAMP in 3T3 cells. Cholera toxin stimulated mitogenesis could not be mimicked by choleragenoid, the binding but inactive subunit of cholera toxin, or by other agents which elevate cAMP levels in 3T3 cells.     

Effects of Detergents on in vitro Growth of Diatom Alga Thalassiosira pseudonana

The growth of the diatom alga Thalassiosira pseudonana was studied when exposed to an environment polluted by a detergent. We determined concentrations that inhibit cell division (10 mg/l) instead of algae growth (0.1 and 1 mg/l. It was shown that T. pseudonana can adapt to high detergent concentrations. The stimulation of the growth of Thalassiosira within a range of 0.03–0.08 mg/l concentration has been registered.     

Characterization of the alterations of the chlorophyll a fluorescence induction curve after addition of Photosystem II inhibiting herbicides

The effects of Photosystem II inhibiting herbicides, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), atrazine and two novel 2-benzylamino-1,3,5-triazine compounds, on photosynthetic oxygen evolution and chlorophyll a fluorescence induction were measured in thylakoids isolated from Chenopodium album (wild type and atrazine-resistant plants) and cyanobacterial intact cells. The resistant plants have a mutation of serine for glycine at position 264 of the D1 protein. Diuron and two members of a novel class of 2-benzylamino-1,3,5-triazine compounds were almost as active in wild-type as in atrazine-resistant thylakoids, indicating that the benzylamino substitution in the novel triazines may be important for the lack of resistance in these atrazine-resistant plants. The inhibition by the herbicides of oxygen evolution in the cyanobacteria was somewhat lower than in the thylakoids of Chenopodium album wild type, probably caused by a slower uptake in the intact cells. The so-called OJIP fluorescence induction curve was measured during a one second light pulse in the absence and in the presence of high concentrations of the four herbicides. In the presence of a herbicide we observed an increase of the initial fluorescence at the origin (Fo′), a higher J level, and a decreased steady state at its P level (Fp). The increase to Fo′ and the decreased leveling Fp are discussed. After dark adaptation about 25% of the reaction centers are in the S₀ state of the oxygen evolving complex with an electron on the secondary electron accepting quinone, Q_B. The addition of a herbicide causes a transfer of the electron on Q_B to the primary quinone acceptor, Q_A, and displacement of Q_B by the herbicide; the reduced Q_A leads to a higher Fo′. The decrease of Fp in the presence of the herbicides is suggested to be caused by inhibition of the photo-electrochemical stimulation of the fluorescence yield. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of the width of the herbicide strip on mite dynamics in apple orchards

Herbicide strips are used in apple orchards to promote tree growth and survival, to increase yield and to reduce the risk of rodent damage to tree bark. However, herbicide strips, particularly wider ones, may cause problems including soil erosion, reduced organic matter, leaching of nitrates into ground water and increased incidence of plant diseases and pests, including two-spotted spider mites, Tetranychus urticae Koch. In this 2 year study we monitored mite dynamics in apple trees and used sticky bands on tree trunks to determine rates of T. urticae immigration into Nova Spy apple trees in plots with wide (2 m) or narrow (0.5 m) herbicide strips. Use of wider herbicide strips promoted two risk factors that could trigger outbreaks of tetranychid mites. First, concentrations of leaf N in apple trees were higher and those of P and K were lower with the wide strips. Such changes in nutritional quality of leaves would increase the potential for more rapid population growth of T. urticae, and to a lesser extent, the European red mite, Panonychus ulmi (Koch). Second, there were higher rates of T. urticae immigration from the ground cover vegetation into the trees. In 2006, and for most of 2007, densities of T. urticae were higher with wide herbicide strips, whereas densities of P. ulmi were not enhanced. However, by late August to early September in 2007, densities of both tetranychids were lower with wide herbicide strips. This is because both risk factors were counterbalanced, and eventually negated, by the enhanced action of phytoseiid predators, mostly Typhlodromus pyri Scheuten. From July through September 2006, ratios of phytoseiids to tetranychids were always several-fold lower with wide herbicide strips but in 2007, from mid-July onwards, predator–prey ratios were usually several-fold higher with wide strips. However, this numerical response of phytoseiids to prey density can only occur where the pesticide program in orchards is not too harsh on phytoseiids. Hence the impact of width of herbicide strip is contingent on the composition and size of the phytoseiid complex and the impact of pesticides on predation.     

Superoxide,hydrogen peroxide,and the respiratory burst of fungally infected plant cells

The principal route of oxygen utilization in the respiratory burst of fungally infected plants was determined from stoichiometries of the uptake and electronic reduction of oxygen in cotton cells exposed to Aspergillus favus walls. Using 2,2-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid) and epinephrine as redox reagents to manipulate oxygen transitions, we found that oxygen consumption doubled when superoxide disproportionation was abolished and was abolished when disproportionation doubled. Of four possible pathways for oxygen consumption, only monovalent reduction of molecular oxygen to superoxide was consistent with this inversely proportional relationship. According to the observed rate of oxygen consumption in this pathway and in the absence of competition to disproportionation of superoxide, infected cells are capable of generating intracellular concentrations of 1 M hydrogen peroxide in 13 min.     

Ultrastructural and biochemical changes in Mycobacterium rubrum and Streptomyces bacillaris cells exposed to the herbicide semeron

The work was aimed at studying how the herbicide semerone affected the ultrastructure of two soil microorganisms, Mycobacterium rubrum and Streptomyces bacillaris. Depending on its concentration, the herbicide inhibited growth processes so that biomass yield decreased, cell division was interfered with, and giant and misshapen cells appeared. The herbicide taken at a concentration of 50-100 mg/ml increased the amount of membrane structures of the respiration type in some cells. This compound at a concentration of 400-500 mg/ml changed the nucleoid structure in certain cells. The decrease number of ribosomes and their peculiar distribution in the cell cytoplasm are most typical responses of the cells to the herbicide action. These responses were found in all cells at any of the tested herbicide concentrations. The results of cytological experiments are supported by statistically reliable data on the effect of the herbicide on RNA and protein synthesis. RNA synthesis is inhibited at a semerone concentration as low as 1 mg/ml, which is a very sensitive indicator of its presence in the medium.     

Selectivity of diclofop-methyl between wheat and wild oat: growth and herbicide metabolism

Abstract Growth of the second leaf of susceptible wild oat (Avena fatua L.) was inhibited within 2 days after treatment with the herbicide, diclofop-methyl, in the 1-1/2 leaf stage. Leaf growth of resistant wheat (Triticum aestivum L.) was unaffected by diclofop-methyl. In wild oat. chlorosis developed 1 day after leaf growth was inhibited. Foliar absorption of diclofop-methyl was similar between wild oat and wheat with 67 and 61% of the recovered radioactivity from [¹⁴C]diclofop-methyl being absorbed by wild oat and wheat, respectively, after 4 days. Wild oal was equally sensitive to the methyl ester and acid forms of the herbicide when the compounds were injected into the stem. Wheat was unaffected by both forms when treated similarly. Very little diclofop-methyl and diclofop (combined total of 10 to 12% in wild oat and 5 to 7% in wheat) remained in plant tissues 2 days after leaf treatment in both susceptible and resistant plants. Therefore, the active form of the herbicide must inhibit growth of susceptible plants very rapidly and at relatively low concentrations. Diclofop-methyl was rapidly hydrolyzed to diclofop by wild oat and wheat. Wild oat predominantly conjugated diclofop to an ester conjugate but wheat hydroxylated the 2,4-dichlorophenyl ring and formed a phenolic conjugate. The formation of the different conjugates between wild oat and wheat was the most significant difference in metabolism between the two species. Nearly 60 and 70% of the methanol-soluble radioactivity was present as water-soluble conjugates in wild oat and wheat, respectively, 4 days after treatment.     

Sensitivity of the Algal Biotest ISO 10253 to the Photosystem 2 Herbicides in Seawater

The sensitivity of marine algal biotest ISO 10253 to the photosystem 2 (PS2) herbicide diuron (DCMU) was determined. Using the diatom Phaeodactylum tricornutum, we found that the algal growth rate was reduced to 50 % of the control value (EC₅₀) for ca. 200 nM DCMU. This value is too high to allow a practical application of the biotest for concentrations of the PS2 herbicides found in natural waters. The mechanisms causing the low sensitivity of the biotest to the PS2 herbicide were investigated by measuring parameters of photosynthetic apparatus in the diatom prior and during the biotest. The apparent dissociation constant for DCMU in P. tricornutum found by measurements of inhibition of oxygen evolution and of variable fluorescence was in the range 60–90 nM. This should lead to a much higher sensitivity of the biotest than found in our experiments. The low biotest sensitivity is caused by an acclimation to sub-lethal DCMU concentrations. The acclimation is manifested by the chlorophyll content per cell that is increasing with the DCMU concentration. During a prolonged exposure to sub-lethal herbicide concentrations, we observed also a selection of DCMU resistant organisms indicating that also an adaptation may decrease the test sensitivity. The biotest sensitivity may increase when the acclimation and adaptation are limited by shortening of the experiment duration. This revised version was published online in August 2006 with corrections to the Cover Date.     

Environmental actions of agrochemicals 1. Side-effects of the herbicide 3-amino-1,2,4-triazole on a laboratory acarine/host-plant interaction (Tetranychus urticae/Phaseolus vulgaris) as revealed by electron microscopy

Foliar and soil application in concentrations below the recommended rate of the herbicide 3-amino-1,2,4-triazole to the host plantPhaseolus vulgaris L. results in structural alterations of the protein-synthesizing apparatus of midgut and salivary-gland cells of the phytophagous spider miteTetranychus urticae Koch (Acari: Tetranychidae) independent of its mode of application. With prolonged incubation times cytological defects become more intense, and spread to more cells and tissues. Resultant effects on yolk and egg formation were expressed as an inhibition of egg deposition that led to a decrease in the reproduction rate ofT. urticae.Consequences of 3-amino-1,2,4-triazole action onT. urticae are discussed with regard to its value to a host-plant/parasite model, agricultural practices and environmental impacts.     

Factors inducing transition from growth to dormancy in rhizobacteria Azospirillum brasilense

The factors suppressing division of the cells of the rhizobacterium Azospirillum brasilense and inducing their transition to a dormant state were analyzed. These included the presence of hexylresorcinol or heavy metals (Cu and Co) in the medium, oxygen stress, and transfer of the cells into the physiological saline or phosphate buffer solution. The results were used to develop a protocol for obtaining of nonculturable cells of A. brasilense Sp245, a natural symbiont of wheat. The cells lost their ability to grow on synthetic agar medium, but could revert to growth when incubated in freshly prepared liquid medium. Needle-shaped crystals differing from struvite, which has been previously reported for this strain, were found in the dormant culture of A. brasilense Sp245.     

Thymus-derived peripheral lymphocytes from human newborns inhibit division of their mothers' lymphocytes.

Lymphocytes from human newborns inhibit division of their mothers' lymphocytes. Three days after we cultured equal numbers of cells from a mother and her baby in the presence of PHA, mitosis of the mother's lymphocytes was suppressed 13-fold compared to that of the baby's lymphocytes. At the end of 3 days the number of baby's lymphocytes were doubled those of the mother's. The survival rates and mean mitotic indexes of both pairs of cell were roughly equivalent (mean +/- S.E: baby 2.4 +/- 0.8; mother 2.6 +/- 0.7), indicating that the lack of dividing lymphocytes from the mother was caused by inhibited division of the mother's lymphocytes, no enhanced growth of the newborn's cells. The cell population in newborns that is responsible for the inhibition effect resides in the T cell-enriched population. Lymphocytes from one newborn were not able to inhibit division of lymphocytes from another newborn, suggesting that lymphocytes from newborns could continue to divide despite their inhibitory effect. Other experiments showed that actively dividing fetal fibroblasts, amnion cells from the newborn, and continuous T lymphoblastoid cell lines were unable to inhibit mitosis of lymphocytes of the mother.     

Inhibition of Cell Migration and Cell Division Correlates with Distinct Effects of Microtubule Inhibiting Drugs

Drugs that target microtubules are thought to inhibit cell division and cell migration by suppressing dynamic instability, a “search and capture” behavior that allows microtubules to probe their environment. Here, we report that subtoxic drug concentrations are sufficient to inhibit plus-end microtubule dynamic instability and cell migration without affecting cell division or microtubule assembly. The higher drug concentrations needed to inhibit cell division act through a novel mechanism that generates microtubule fragments by stimulating microtubule minus-end detachment from their organizing centers. The frequency of microtubule detachment in untreated cells increases at prophase suggesting that it is a regulated cellular process important for spindle assembly and function. We conclude that drugs produce differential dose-dependent effects at microtubule plus and minus-ends to inhibit different microtubule-mediated functions.     

An analysis of the recovery of tetrahymena from effects of cycloheximide

When cycloheximide (0.2 μg per ml) was added to synchronized cultures of Tetrahymena pyriformis GL-C, the initial rate of incorporation of ¹⁴C-leucine was reduced to about 20% of the rate observed in control cells. After one hour, the rate increased fairly abruptly to about 60% of the control rate. The cells in cycloheximide underwent synchronous division about three hours after addition of cycloheximide. A second addition of cycloheximide had little effect on either the rate of incorporation or on the time of cell division in the drug. The medium in which cells had recovered brought about full inhibition of ¹⁴C-leucine incorporation in fresh cells, indicating that recovery was not accompanied by appreciable degradation of the cycloheximide. It was therefore concluded that during recovery the cells were either adapting to the cycloheximide or excluding it. The recovery process shows some specificity, since cells which had recovered from cycloheximide, and had become insensitive to a second dose of this drug, still retained full sensitivity to another drug, colchicine. Conversely, cells recovering in colchicine became insensitive to fresh colchicine but remained sensitive to cycloheximide.     

Mass cultivation of Tetrahymena thermophila yielding high cell densities and short generation times

Summary A cheap medium, composed of skimmed milk powder, yeast extract, and glucose, for mass cultivation of the protozoon Tetrahymena thermophila has been developed. Cell concentrations of 5 x 10⁶ cells/ml and unprecedented short generation times of only 1.4 h were determined in batch cultures. During the exponential phase of growth, daughter cells initiated a new cell division even before the previous division had been completed, resulting in the formation of cell chains. Addition of glucose extended the stationary phase. Using a bench-top fermentor supplied with a digital control unit the utilization of nutrient components in batch culture was monitored. Milk protein and glucose were consumed completely, but lactose only partly. Correspondence to: A. Tiedtke     

Toxic effects of fatty acids on yeast cells: dependence of inhibitory effects on fatty acid concentration.

The yeasts Saccharomyces cerevisiae, Candida utilis, and Candida lipolytica were used to investigate the action of different concentrations of fatty acids (from acetic to myristic acid) on cell growth, division, uptake of inorganic phosphate, and substrate oxidation. The former two yeasts were found to undergo an inhibition of growth, cell division, and phosphate uptake at lower acid concentrations and to experience the inhibition of substrate oxidation at higher acid concentrations. The concentration dependence of the action of fatty acids can be classified into four categories: 1) subthreshold concentrations which do not inhibit growth and have either no effect on, or stimulate, oxygen consumption; 2) threshold concentrations which lower the rate of growth, cell division, and phosphate uptake but do not inhibit the oxidation of carbon substrate; 3) above-threshold concentrations which inhibit partially even substrate oxidation, and 4) microbicide concentrations. Candida lipolytica displays the same sensitivity toward the action of fatty acids as the above yeast species; however, the threshold concentrations are higher and can be quickly lowered owing to oxidation by the yeast. The concentrations of fatty acids found in the medium after cultivations of yeast with n-alkanes are of the same order as limiting concentrations; the formation of acids with twelve and less carbons in the molecule can thus be assumed to be one of the basic reasons for lowering of biomass yields during cultivations on these hydrocarbons.     

Kinetics of microbial oxidation of n-heptane. I. Characterization of the process

Utilization of n-heptane by a Pseudomonad was studied in pilot-size butch cultures. Optimal pH and temperature were determined by a factorial design and a medium based upon mineral uptake rates was formulated. High cell yields were obtained by volatilizing heptane in the incoming air and thereby achieving good hydrocarbon dispersion. Hydrocarbon carried by effluent gases was recovered and recycled. In cultures where pH is not controlled, decrease in the electrolytic conductivity of the medium was found to be indicative of viable cells and was used in monitoring bacterial propagation. If not checked, increase in salinity in pH controlled cultures was found to affect cell production negatively. Viscosity changes were not very significant. Heptane to aqueous medium ratio was found to affect oxygen supply to the system due to higher dissolved oxygen concentrations associated with hydrocarbons.     

Bacterial cell division: regulating Z-ring formation

The earliest stage of cell division in bacteria is the formation of a Z ring, composed of a polymer of the FtsZ protein, at the division site. Z rings appear to be synthesized in a bi‐directional manner from a nucleation site (NS) located on the inside of the cytoplasmic membrane. It is the utilization of a NS specifically at the site of septum formation that determines where and when division will occur. However, a Z ring can be made to form at positions other than at the division site. How does a cell regulate utilization of a NS at the correct location and at the right time? In rod‐shaped bacteria such as Escherichia coli and Bacillus subtilis, two factors involved in this regulation are the Min system and nucleoid occlusion. It is suggested that in B. subtilis, the main role of the Min proteins is to inhibit division at the nucleoid‐free cell poles. In E. coli it is currently not clear whether the Min system can direct a Z ring to the division site at mid‐cell or whether its main role is to ensure that division inhibition occurs away from mid‐cell, a role analogous to that in B. subtilis. While the nucleoid negatively influences Z‐ring formation in its vicinity in these rod‐shaped organisms, the exact relationship between nucleoid occlusion and the ability to form a mid‐cell Z ring is unresolved. Recent evidence suggests that in B. subtilis and Caulobacter crescentus, utilization of the NS at the division site is intimately linked to the progress of a round of chromosome replication and this may form the basis of achieving co‐ordination between chromosome replication and cell division.