Micronucleation by mitosis inhibitors in developing microspores of Spathiphyllum wallisii Regel.

KEY MESSAGE : We developed an efficient protocol for chromosome scattering in Spathiphyllum microspores. The effects of plant material, developmental age, genotype and antimicrotubular toxin type, exposure and concentration were evaluated. Asymmetric hybridization through microprotoplast-mediated chromosome transfer (MMCT) is a known method for overcoming sexual breeding barriers between distantly related plant species. To obtain microprotoplasts, it is necessary to induce mass micronucleation either in somatic or gametic cells. We have tested the efficiency for micronuclei induction of five mitosis inhibitors, amiprophos-methyl (APM), butamiphos (BUT), chlorpropham (CIPC), oryzalin (ORY) and propyzamide (PRO), on developing microspores of diploid Spathiphyllum wallisii Regel. Besides the used toxins, also the effect of their concentrations and incubation period as well as plant genotypes and material was tested. We observed micronuclei (MNi) in pollen mother cells, dyads and tetrads as well as other abnormalities such as ball metaphases and chromosome bridges. The flower position on the spadix and the type of starting material (dissected anthers vs. complete spadices) did not significantly influence micronucleation frequencies. The highest micronucleation index of 86 % was obtained in microspores treated with 10 µM ORY during 72 h. All six genotypes tested formed micronuclei after this particular treatment, although the efficiency varied between cultivars. Next to ORY, CIPC was also a very efficient MNi inducer. The average number of MNi found in micronucleated cells varied between 1.67-6.44 for CIPC and 0.83-5.50 for ORY. The maximal number of MNi observed was 12 for CIPC and 9 for ORY. Our results demonstrate that CIPC and ORY can be applied for mass micronucleation on developing microspores of S. wallisii as a first step of MMCT in aroid interspecific or intergeneric breeding.

The use of bio-guided fractionation to explore the use of leftover biomass in Dutch flower bulb production as allelochemicals against weeds.

A major problem in flower bulb cultivation is weed control. Synthetic herbicides are mainly used, although they cause a range of problems, and integrated weed control through application of naturally occurring allelochemicals would be highly desirable. Flower bulb production creates large amounts of leftover biomass. Utilizing this source for weed control may provide new applications of the bulb crops. We therefore screened 33 flower bulb extracts for allelochemical activity against weeds. Several methanol and chloroform extracts were observed to inhibit germination and growth of Senecio vulgaris L. and Lolium perenne L., as representatives of di- and mono-cotyledonous weeds, respectively. Narciclasine was identified as the bioactive compound in Narcissus. The extract of Amaryllis belladonna L. was equally active, but did not contain any narciclasine. Bioassay-guided fractionation of the A. belladonna extract resulted in the identification of lycorine as the bio-active compound. The IC50 measured for radicle growth inhibition was 0.10 µM for narciclasine and 0.93 µM for lycorine, compared to 0.11 mM of chlorpropham, a synthetic herbicide. Therefore, the leftover biomass from the spring bulb industry represents an interesting potential source for promising allelochemicals for further studies on weed growth inhibition.

Carbohydrate metabolism dynamic in chlorpropham- and 1,4-dimethylnaphthalene-treated potatoes and its effect on the browning of French fries.

The use of a sprout suppressor is crucial for the use of potatoes beyond their natural dormancy period. The main sprout inhibitor used on a commercial scale, chlorpropham (CIPC), is becoming increasingly limited owing to its toxicity. Therefore, we evaluated the effectiveness of 1,4-dimethylnaphthalene (1,4-DMN) compared to CIPC in controlling sprouting and maintaining the quality of potato, Solanum tuberosum 'Asterix', during cold storage. Treatment with 1,4-DMN reduced fresh weight loss and controlled the number and length of sprouts comparable to CIPC. Compared to the control, both sprouting inhibitors led to higher starch and lower reducing sugar contents, and the tubers retained the recommended quality for industrial processing. After frying, less browning was observed in French fries obtained from 1,4-DMN- or CIPC-treated tubers. We ascertain that 1,4-DMN besides being an efficient sprouting inhibitor and alternative to CIPC, it contributes to maintaining the quality of French fries after cold storage.

The sprout inhibitors chlorpropham and 1,4-dimethylnaphthalene elicit different transcriptional profiles and do not suppress growth through a prolongation of the dormant state.

Chlorpropham (CIPC) and 1,4-dimethylnapthalene (DMN) are used to control postharvest sprouting of potato tubers. CIPC alters microtubule structure and function resulting in inhibition of cell division. The mechanism of action of DMN is unknown but, because it is a natural product found in potato tubers, there is speculation that it inhibits sprout growth by prolonging the dormant state. To address this issue, the effects of CIPC and DMN on abscisic acid (ABA) content and gene expression in potato tuber meristems were determined and compared to those found in dormant and non-dormant meristems. Dormancy progression was accompanied by a dramatic decline in ABA content and the ABA levels in meristems isolated from CIPC- and DMN- treated tubers were identical to the levels found in nondormant meristems demonstrating that sprout repression is not a function of elevated ABA. Evaluation of transcriptional profiles using cDNA microarrays demonstrated that there were similarities between CIPC- and DMN- treated tuber tissues particularly in transcripts that encode phosphatases and proteins associated with oxygen-related metabolism. Despite these similarities, there were significant differences in transcript profiles derived from treatment with either CIPC or DMN and the dormant state. These results suggested the mechanisms-of -action of DMN and CIPC are distinct and not due to a prolongation of the normal dormant condition.

Microtubule biogenesis and cell shape in Ochromonas. 3. Effects of herbicidal mitotic inhibitor isopropyl N-phenylcarbamate on shape and flagellum regeneration.

The role of microtubules and microtubule nucleating sites in the unicell, Ochromonas has been examined through the use of two mitotic inhibitors, isopropyl N-phenylcarbamate (IPC) and isopropyl N-3-chlorophenyl carbamate (CIPC). Although IPC and CIPC have little or no effect on intact microtubules, the assembly of three separate sets of microtubules in Ochromonas has been found to be differentially affected by IPC and CIPC. The assembly of flagellar microtubules after mechanical deflagellation is partially inhibited; the reassembly of rhizoplast microtubules after pressure depolymerization is totally inhibited (however, macrotubules may form at the sites of microtubule initiation or elsewhere); and, the reassembly of the beak set of microtubules after pressure depolymerization may be unaffected although similar concentrations of IPC and CICP completely inhibit microtubule regeneration on the rhizoplast. These effects on microtubule assembly, either inhibitory or macrotubule inducing, are fully reversible. The kinetics of inhibition and reversal are found to be generally similar for both flagellar and cell shape regeneration. Incorporation data suggest that neither IPC nor CIPC has significant effects on protein synthesis in short term experiments. Conversely, inhibiting protein synthesis with cycloheximide has little effect on microtubule regeneration when IPC or CIPC is removed. Although the exact target for IPC and CIPC action remains uncertain, the available evidence suggests that the microtubule protein pool or the microtubule nucleating sites are specifically and reversibly affected. Comparative experiments using the mitotic inhibitor colchicine indicate some similarities and differences in its mode of action with respect to that of IPC and CIPC on assembly and disassembly of microtubules in these cells.

The relationship between the division plane and spindle geometry in Allium cells treated with CIPC and griseofulvin: an anti-tubulin study.

Isopropyl N-(3-chlorophenyl)-carbamate (CIPC), and griseofulvin, were used to perturb mitosis and the subsequent plane of division in meristematic cells of Allium cepa. The effects of these compounds on the microtubule organization throughout the cell cycle were investigated by immunofluorescence techniques. Microtubules were not disassembled by drug treatment, but the spindle organization was disrupted, resulting in tripolar spindles which gave rise to multiple nuclei. Ensuing cell plates, with associated phragmoplast microtubules, were branched. The effects of these drugs with respect to MTOC duplication and function in plant cells are discussed as is the relationship between the pre-prophase band (PPB) and the plane of cell division.

Na+-coupled D-glucose uptake and membrane order of enterocyte brush border membrane vesicles, under the effect of a series of N-phenylcarbamates.

The importance of the hydrophobic effect of exogenous substances and of modifications of membrane order on D-glucose uptake are still poorly defined. Our results show that the concentrative Na+ -coupled D-glucose uptake of rat enterocyte brush border membrane vesicles is inhibited by N-phenylcarbamates increase the membrane order. However, since the concentrations required for membrane order increase are much greater than those active on D-glucose uptake, the effects on lipid order cannot be responsible for the inhibition of D-glucose uptake. Measurements of D-glucose uptake under conditions of Na+ equilibrium show that these carbamates do not act directly on the carrier but indirectly by favouring the dissipation of the Na+ gradient.

Anti-herbicide single-chain antibody expression confers herbicide tolerance in transgenic plants.

An anti-chlorpropham single-chain variable-fragment (scFv) gene was introduced into Arabidopsis in a manner to express the antibody fragment in each of four different subcellular compartments. The accumulation of scFv in transgenic plants was detected by targeting the fragment in the endoplasmic reticulum or apoplastic space, or by expressing the fragment as a glycosylphosphatidylinositol-anchored protein, while no accumulation could be detected by targeting the fragment in the cytosol. Transgenic plants accumulating the scFv gene at a high level in the endoplasmic reticulum had enhanced tolerance to chlorpropham in comparison with the non-transformants.

Effects of chlorpropham (CIPC) on the hemopoietic system of rats.

Male F344 rats were given 0 or 3% chlorpropham in the diet and at 2, 4, 6, 8 or 13 weeks of administration, five rats in each group were examined for hematology, plasma clinical chemistry and pathology. Marked splenomegaly and hepatomegaly were observed in treated rats at 2-13 weeks of administration. Red blood cell counts, hemoglobin concentration, packed cell volume and platelet counts were significantly decreased and methemoglobin level, mean corpuscular volume and white blood cell counts were significantly increased in treated rats at 2-13 weeks of administration. The covalent binding of m-chloraniline m-CA, (the hydrolytic metabolite of chlorpropham) was observed in hemoglobin or splenic protein of treated rats, but only small amounts of free m-CA were present in blood or spleen. Congestion, hemosiderin deposits, extramedullary hemopoiesis and lymphoid atrophy in spleen and hyperplasia of hemopoietic cells in bone marrow were observed in treated rats at 2-13 weeks and fibrosis in splenic capsule were observed in treated rats at 4-13 weeks. The pathological changes in spleen rather than hematological changes progressed during administration, suggesting splenotoxicity of CIPC in rats.

Immunofluorescence microscopy of microtubules in intact cell lineages of the moss, Physcomitrella patens. I. Normal and CIPC-treated tip cells.

Monoclonal antibodies to yeast tubulin have been used to visualize the distribution of microtubules in the intact filamentous protonemata of the moss Physcomitrella patens. Protonemata were prepared for immunofluorescence by fixation in formaldehyde and cells were made permeable with Driselase. Extensive cell files were preserved by 'blotting' the moss onto glutaraldehyde-derivatized coverslips. Problems due to fluorescence from chloroplasts were obviated by extraction with dimethyl sulphoxide and the non-ionic detergent, Nonidet NP40. These improvements allowed us to determine that microtubules were present throughout the cell cycle in the apical dome of caulonemal tip cells, that was a pronounced association of microtubules with the nucleus, that 'astral' microtubules were associated with the mitotic spindle and during anaphase may be involved in reorientation of the spindle before an oblique cytokinesis in caulonemata and that the cytokinetic phragmoplast appeared identical to the structure described for higher plants. Microtubules appeared to converge at the very tip of apical caulonemal cells and this was studied further by treating cells with CIPC--a drug that is known to produce multiple microtubule-organizing centres--and which here produces multiple foci for microtubules at the tip. These observations emphasize the involvement of microtubules in tip growth, alignment of the cell plate and nuclear migration--processes that are fundamental to the morphogenesis of filamentous organisms.

Isolation and characterization of mutants supersensitive to the spindle poison, isopropyl N-3-chlorophenyl carbamate (CIPC) in the fission yeast Schizosaccharomyces pombe.

Mutants supersensitive to the spindle poison, Isopropyl N-3-chlorophenyl carbamate (CIPC) of the fission yeast Schizosaccharomyces pombe were isolated and characterized genetically. Fourteen different recessive loci were assigned for the mutation (donated as cps1 to cps14) and two, cps1 and cps3, were mapped precisely on the chromosomes. Nine mutant strains were also supersensitive to phenothiazine derivatives, inhibitors of calcium-binding protein calmodulin. Four of nine strains were incapable of growing in the presence of 10 microM calcium ionophore A23187, at which the drug had no effect on cell growth in other strains. Fluorescence microscopy using the DAPI and Calcofluor staining methods showed two strains out of four to be defective in normal cell division; most stationary-phase cells of the cps6 mutant were seen to be bi- or tetra-nucleate, being partitioned with one or three septa, respectively. In the other mutant (cps8), enlarged cells were unequally partitioned with multisepta, and each compartment contained several daughter nuclei. The septa appeared aberrant in position within the cell, and situated diagonally but not vertically along the long cell axis.

Study of potatoes' sprout inhibitor treatments with chlorprophame.

Studies carried out in 1999 by the University of Ghent showed that 36% of potatoes' samples contained Chlorprophame (CIPC) residues and that 7.9% of them exceeded the maximal limit of residues (RML), fixed at 5 ppm. The heterogeneity of sprout inhibitor application would be one of the causes of over-dosage. However, this heterogeneity would also cause under-dosages leading to problems when controlling the sprouting in potatoes stored over 6 degrees C. This study aims at determining some technical causes of the heterogeneity of CIPC sprout inhibitor treatments when storing potatoes. The study concerns two treatment techniques: dusting and spraying. To draw up an inventory of mechanical treatments in Belgium, a survey has been conducted among 28 farmers throughout Belgium. 35 samples have been taken at random in the different storage rooms to analyse the content of CIPC residue. In order to do so, a method of analysis: the gas chromatography in capillary phase with detection by mass spectrophotometry, has been developed. Tests have been carried out by changing several parameters such as the material, the product or the place in the storage line, in order to assess the CIPC application techniques. The survey made it possible to analyse qualitatively, from the declarations of farmers, the causes of heterogeneity linked to treatment techniques. An almost systematically over-dosage of the CIPC quantity has been noticed. However, out of the 35 samples analysed, only 2 had residue contents higher than the RML. The comparative analysis of the quantities applied and the residues contained in the samples made it possible to quantify the heterogeneity of the applications depending on the techniques. The tests carried out show in a general way that mechanical dusting, even though having a less constant flowrate than sprayers, leads to less important variation of the residue between samples. In testing conditions, the heterogeneity of the antigerminative treatment decreases when applied by means of a mechanical duster. In practise, these results are distorted by topical applications of CIPC. The combination of this practise with a too high heterogeneity of the treatment are to be avoided in order to have a good preservation and meet the residues standards.

[Effects of propham and chloropropham on mitosis in cultured human lymphocytes. Ultrastructural study]. / Action du propham et du chloropropham sur la mitose de lymphocytes humains en culture. Etude ultrastructurale.

Propham and Chloropropham disturb the several mitotic figure components of proliferating human lymphocytes. The tubular structures, affected in preference by the C.I.P.C., become abnormal, disorganized or disappear. The chromosomal changes (protuberances,absence of condensation and chromosomal bridges) are principally given by I.P.C. I.P.C. and C.I.P.C. lead to the disappearance of the endoplasmic reticulum. These effects are correlated with the action of the two pesticides on DNA and protein synthesis.

The role of the cytoskeleton in the polarized growth of the germ tube in Candida albicans.

Cells of the dimorphic yeast Candida albicans are easily induced to germinate in synchrony. Using germinating cells of strain FC18, we examined the effects of several drugs that are known to affect the cytoskeleton on growth and cytoskeletal organization. Cytochalasin A (CA), an inhibitor of actin function, inhibited the germination of the yeast cells and changed the cylindrical expansion of the apex of the germ tube to swelling growth. Effects of CA on the organization of actin were examined with rhodamine-phalloidin (Rh-Ph), which specifically stains F-actin. In CA-untreated cells, Rh-Ph staining resulted in condensed dot-like fluorescence at the growing tip, as well as filamentous fluorescence (actin cables) that ran from the apex to the basal region. In CA-treated cells, condensed dot-like fluorescence was still observed at the swelling tip, but actin cables had disappeared completely. This result indicates that CA does not affect the asymmetrical distribution of actin, and suggests that the actin cables are not required for maintenance of the polarized localization of actin. Benomyl, an anti-microtubule drug, inhibited the germination of yeast cells and the apical growth of germinated cells. Benomyl not only disrupted microtubules (MTs), but also affected the distribution of actin. In benomyl-treated cells, actin dots were randomly dispersed all over the cell. This result indicates that benomyl destroyed the mechanism that maintains the asymmetrical distribution of actin, and suggests that MTs are involved in such a mechanism. The polarized localization of organelles is one of the most important factors associated with dimorphism.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of microfilaments and microtubules in apical growth and dimorphism of Candida albicans.

Cytoskeleton inhibitors were used to study morphogenesis in the pathogenic and dimorphic fungus Candida albicans. Nocodazole is a specific microtubule inhibitor and chloropropham (CIPC), at high concentrations, is an inhibitor of microtubules and microfilaments. Distribution of microtubules and microfilaments was studied by immunofluorescence techniques using anti-tubulin antibody with FITC-conjugated secondary antibody, and by staining with Rh-phalloidin. Nocodazole did not arrest apical cell elongation at a concentration (20 micrograms ml-1) that inhibited nuclear division and migration. Cytoplasmic and nuclear microtubules disappeared within 30 min in filamentous cells under these conditions. However, the Rh-phalloidin-stained actin granules which were localized in the tips of filamentous cells, and the microfilaments, were arranged normally at this concentration of nocodazole. Growth, and normal distribution of microtubules and microfilaments, were inhibited by a high concentration (200 micrograms ml-1) of CIPC. At a concentration (100 micrograms ml-1) of CIPC that permitted nuclear division, apical cell elongation was arrested, and filamentous growth was converted into yeast growth. At this concentration of CIPC, microtubules were distributed normally in filamentous cells. Long microfilaments were not observed, and actin granules did not localize in the tips of filamentous cells, but were distributed throughout the cytoplasmic cortex. Our results show that cytoplasmic microtubules are not essential for the elongation of filamentous cell tips but that microfilaments are apparently essential for this process.

Biotransformation of chlorpropham (CIPC) in isolated rat hepatocytes and xenoestrogenic activity of CIPC and its metabolites by in vitro assays.

1: The metabolism and action of chlorpropham (isopropyl N-(3-chlorophenyl)carbamate; CIPC, a post-harvest agent) were studied in freshly isolated rat hepatocytes, and the oestrogen-like activity of CIPC and its metabolites was assessed by in vitro assays. The exposure of hepatocyte suspensions to CIPC caused concentration- (0.25-1.0 mM) and time- (0-3 h) dependent cell death, which was assessed by Trypan blue exclusion, accompanied by losses of cellular adenosine triphosphate and adenine nucleotide pools, and formation of cell bleb. 2: CIPC at a weakly toxic level (0.25 or 0.5 mM) was metabolized to isopropyl N-(3-chloro-4-hydroxyphenyl)carbamate (4OH-CIPC) and subsequently to its glucuronide and sulfate conjugates (major metabolites) or alternatively to the minor metabolites 3-chloroaniline (3CA) and 3-chloroacetanilide. CIPC (0.25 mM) added to hepatocyte suspensions was distributed equally between hepatocytes and the extracellular medium during the incubation. The glucuronide rather than the sulfate conjugate of 4OH-CIPC predominantly increased in the medium with time, while the amount of unconjugated free 4OH-CIPC in the extracellular medium increased by approximately threefold compared with the amount in the cell fraction after 0.5 h and then decreased rapidly accompanied by increases in the conjugates. This indicates that unconjugated free 4OH-CIPC produced in hepatocytes was temporarily excreted in the extracellular medium and subsequently converted to the conjugates via re-influx into hepatocytes. 3: Diethylstilbestrol (DES), bisphenol A (BPA) and 4-hydroxybenzoic acid butyl ester (butylparaben), which are known xenoestrogenic compounds, competitively displaced 17beta-oestradiol bound to the oestrogen receptor-alpha (ERalpha) in a concentration-dependent manner; IC50 values of DES, BPA, butylparaben and its derivative 3-chloro-4-hydroxybenzoic acid butyl ester (3-chloro-butylparaben) were approximately 10(-8), 10(-5), 5 x 10(-5) and 5 x 10(-4) M, respectively. In contrast, neither CIPC nor 4OH-CIPC impaired the binding of 17beta-oestradiol to ERalpha at concentrations ranging from 10(-9) to 10(-4) M, whereas at concentrations of >5 x 10(-4) M, the binding affinity of 4OH-CIPC was greater than that of CIPC. In a proliferation assay of MCF-7 cells, CIPC, 4OH-CIPC and 3CA did not increase cell numbers at concentrations ranging from 10(-9) to 10(-5) M, but these compounds at a concentration of 10(-4) M induced a considerable decrease in cell numbers relative to the control. The results suggest that even if CIPC is metabolized to 4OH-CIPC by hepatocytes, the chlorine adjacent to the 4-hydroxy group added to the intermediate as well as 3-chloro-butylparaben obstructs the appearance of oestrogen-like effects via an interaction between the intermediate and the ER.

Cell cycle specificity of certain antimicrotubular drugs in Schizosaccharomyces pombe.

Of the seven antimicrotubular drugs tested, nocodazole, mebendazole and trifluralin at saturable concentrations failed to inhibit cell division in Schizosaccharomyces pombe, while carbendazim, thiabendazole and chloropropham each at 50 micrograms ml- and amiprophos methyl at 200 micrograms ml-1 completely arrested cell division. This inhibition was associated with striking morphological changes in which carbendazim- and thiabendazole-treated cells became elongated and pseudohyphal, whereas chloropropham- and amiprophos methyl-treated cells appeared small and rounded with occasional V-shaped pairs. Lomofungin staining revealed that nuclear division was also arrested by these drugs. Suspected blockage of defined cell cycle stages was confirmed by pulse-induction experiments which revealed that cells could be synchronized into division using exposure to a drug for one generation. Further experiments with synchronous cultures prepared by size selection showed that different drugs possessed different transition points; for example, carbendazim and thiabendazole were effective in blocking a late stage of the cell cycle just prior to division, whereas amiprophos methyl affected a very early stage. The results suggest that some of the drugs used exert cell cycle specificity in S. pombe either by impairing microtubule assembly mechanisms (as with carbendazim and thiabendazole) or by inhibiting synthesis of tubulin subunits (as with amiprophos methyl). These drugs could prove useful in studies of microtubule biogenesis during the cell cycle in yeast.

Evidence that herbicides relaxing smooth and oblique-striated muscles affect the muscle cell membrane.

1. From the example of two herbicides [chlorpropham (CIPC) and terbutryn], it has been shown that such compounds inhibit spontaneously occurring, as well as pharmacologically (ACh, KCl, caffeine) or electrically (negative DC) evoked tonic and phasic activities in different smooth and oblique-striated muscles of snails [penis retractor muscle (PRM) of Helix], worms (torsos and muscle segments of Lumbricus and Eisenia) and mice (small intestine of Mus). In PRM preparations, experiments with the denervating drugs 6-OHDA, -5,6-DHT, and reserpine, or with the serotonergic receptor blocker methysergide, produced evidence that the herbicidal side-effect was not caused by action on the nervous system. 3. A relaxant effect on ACh-evoked contractions in the PRM was induced by drugs altering the Ca2+ equilibrium, for example, theophylline, papaverine, chlorpromazine, or trifluoperazine. 4. In addition, an extracellular Ca2+ deficiency or the presence of papaverine led to an enhancement of a CIPC-caused inhibition of ACh- or KCl-induced contractions. 5. The amplitudes of chemically evoked contractions in "skinned muscle cells" of Helix PRM or Lumbricus segments were influenced neither by CIPC nor by terbutryn. 6. CIPC was concentrated by the intact PRM and by a membrane containing PRM fraction, as well as by the worms' circular muscle system. 7. A fluorescent CIPC analogue, dansyl-3-chloroaniline, characterized by a similar inhibiting property on the induced contractions was detected at the border of PRM cells. 8. It is concluded that in the different muscle systems the side-effect of the herbicidal compounds is located in the outer muscle cell membrane where a Ca2+-dependent mechanism may be concerned.