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Multipurpose contact lens disinfecting solutions (MPDS) are widely used to cleanse and disinfect microorganisms. However, disinfection efficacy of these MPDS against Acanthamoeba cyst remain insufficient. 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, is capable of increasing the amoebical effect against Acanthamoeba by inhibiting its encystation. In this study, we investigated the possibility of DCB as a disinfecting agent to improve the amoebicidal activity of MPDS against Acanthamoeba cyst. Eight commercial MPDS (from a to h) were assessed, all of which displayed insufficient amoebicidal activity against the mature cysts. Solution e, f, and h showed strong amoebicidal effect on the immature cysts. Amoebicidal efficacy against mature cysts remained inadequate even when the 8 MPDS were combined with 100 μM DCB. However, 4 kinds of MPDS (solution d, e, f, and h) including 100 μM DCB demonstrated strong amoebicidal activity against the immature cysts. The amoebicidal activity of solution d was increased by addition of DCB. Cytotoxicity was absent in human corneal epithelial cells treated with either DCB or mixture of DCB with MPDS. These results suggested that DCB can enhance the amoebicical activity of MPDS against Acanthamoeba immature cyst in vitro.

International audience; Pesticides taken up by plants from the soil or interstitial (pore) water can cascade to higher trophic levels, which are expected to be more affected due to cumulative bottom-up effects. Knowledge about the impact of indirect exposure to pesticides on non-target terrestrial trophic chains, however, is still lacking. Therefore, we examined the direct and indirect effects of three concentrations of the herbicide 2,6-dichlorobenzonitrile (DCBN) and an insecticide with a similar molecular structure (1,4-dichlorobenzene, DCB) on the fitness traits of a tritrophic system: the wheat plant Triticum aestivum, the aphid Sitobion avenae and its specialist parasitoid Aphidius rhopalosiphi. To mimic exposure via interstitial water the toxicants were added to the growth medium of the plant. Passive dosing between the medium and a silicon layer was used to achieve constant exposure of the poorly soluble pesticides. Wheat plants exposed to both pesticides grew smaller and had reduced biomasses. Negative effects on the reproductive rate, biomass and the number of aphids were only observable at the highest concentration of DCBN. Overall parasitism rate decreased when exposed to both pesticides and parasitoid attack rates decreased at lower concentrations of DCBN and at the highest DCB concentration. The parasitoid sex ratio was extremely male-biased in the presence of DCBN. Our results demonstrate that pesticides can alter the performance of higher trophic levels by sublethal effects, through a bypass of the second trophic level. In addition, the novel test system used was suitable for detecting such carryover effects on non-target organisms.

N1-substituted octahydro-1 H-pyrano[2,3- d]pyrimidines derivatives were prepared in moderate yield by the reaction of methyl [(4a RS,5 SR,10a RS)-5-aryl-2-oxo-3,4,4 a,10 a-tetrahydro-2 H,5 H-pyrano[3,2- e][1,3]thiazolo[3,2- a]pyrimidin-8(9 H)-ylidene]acetate and 2,6-dichorobenzonitrile oxide via a domino 1,3-dipolar cycloaddition/ring-opening/dethionation process. The structures of the products were characterised by spectroscopic and X-ray crystallographic analysis.

Summary As a consequence of the habituation to low levels of dichlobenil (DCB), cultured maize cells presented an altered hemicellulose cell fate with a lower proportion of strongly wall-bound hemicelluloses and an increase in soluble extracellular polymers released into the culture medium. The aim of this study was to investigate the relative molecular mass distributions of polysaccharides as well as phenolic metabolism in cells habituated to low levels of DCB (1.5 uM). Generally, cell wall bound hemicelluloses and sloughed polymers from habituated cells were more homogeneously sized and had a lower weight-average relative molecular mass. In addition, polysaccharides underwent massive cross-linking after being secreted into the cell wall, but this cross-linking was less pronounced in habituated cells than in non-habituated ones. However, when relativized, ferulic acid and p-coumaric acid contents were higher in this habituated cell line. Feasibly, cells habituated to low levels of DCB synthesized molecules with a lower weight-average relative molecular mass, although cross-linked, as a part of their strategy to compensate for the lack of cellulose.

The herbicide 2,6-dichlorobenzonitrile (DCBN) is a potent nasal toxicant in rodents; however, it is not known whether DCBN causes similar nasal toxicity in humans. The tissue-selective toxicity of DCBN in mouse nasal mucosa is largely dependent on target tissue bioactivation by CYP2A5. The human orthologues of CYP2A5, CYP2A6 and CYP2A13, are both expressed in nasal mucosa and are capable of activating DCBN. In this study, we directly determined the ability of human nasal mucosa to bioactivate DCBN. We also tested the suitability of a glutathione conjugate of DCBN (GS-DCBN) or its derivatives as biomarkers of DCBN exposure and nasal toxicity in mouse models. We found that human fetal nasal mucosa microsomes catalyze the formation of GS-DCBN, with a Km value comparable to that of adult mouse nasal mucosa microsomes. The activity of the human nasal mucosa microsomes was inhibited by 8-methoxypsoralen, a known CYP2A inhibitor. GS-DCBN and its metabolites were detected in the nasal mucosa and nasal-wash fluid obtained from DCBN-treated mice, in amounts that increased with escalations in DCBN dose, and they were all still detectable at 24 h after a DCBN treatment (at 10 mg/kg). Further studies in Cyp2a5-null mice indicated that GS-DCBN and its metabolites in nasal-wash fluid were generated in the nasal mucosa, rather than in other organs. Thus, our data indicate for the first time that the human nasal mucosa is capable of bioactivating DCBN and that GS-DCBN and its metabolites in nasal-wash fluid may collectively serve as indicators of DCBN exposure and potential nasal toxicity in humans.

Cellulose is the major component of plant cell walls and is an important source of industrial raw material. Although cellulose biosynthesis is one of the most important biochemical processes in plant biology, the regulatory mechanisms of cellulose synthesis are still unclear. Here, we report that 2,6-dichlorobenzonitrile (DCB), an inhibitor of cellulose synthesis, inhibits Arabidopsis root development in a dose- and time-dependent manner. When treated with DCB, the plant cell wall showed altered cellulose distribution and intensity, as shown by calcofluor white and S4B staining. Moreover, pectin deposition was reduced in the presence of DCB when immunostained with the monoclonal antibody JIM5, which was raised against pectin epitopes. This result was confirmed using Fourier transform infrared (FTIR) analysis. Confocal microscopy revealed that the organisation of the microtubule cytoskeleton was significantly disrupted in the presence of low concentrations of DCB, whereas the actin cytoskeleton only showed changes with the application of high DCB concentrations. In addition, the subcellular dynamics of Golgi bodies labelled with N-ST-YFP and TGN labelled with VHA-a1-GFP were both partially blocked by DCB. Transmission electron microscopy indicated that the cell wall structure was affected by DCB, as were the Golgi bodies. Scanning electron microscopy showed changes in the organisation of cellulose microfibrils. These results suggest that the inhibition of cellulose synthesis by DCB not only induced changes in the chemical composition of the root cell wall and cytoskeleton structure, but also changed the distribution of cellulose microfibrils, implying that cellulose plays an important role in root development in Arabidopsis.

We have identified a gene, denoted PttMAP20, which is strongly up-regulated during secondary cell wall synthesis and tightly coregulated with the secondary wall-associated CESA genes in hybrid aspen (Populus tremula × tremuloides). Immunolocalization studies with affinity-purified antibodies specific for PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell walls. This PttMAP20 protein sequence contains a highly conserved TPX2 domain first identified in a microtubule-associated protein (MAP) in Xenopus laevis. Overexpression of PttMAP20 in Arabidopsis (Arabidopsis thaliana) leads to helical twisting of epidermal cells, frequently associated with MAPs. In addition, a PttMAP20-yellow fluorescent protein fusion protein expressed in tobacco (Nicotiana tabacum) leaves localizes to microtubules in leaf epidermal pavement cells. Recombinant PttMAP20 expressed in Escherichia coli also binds specifically to in vitro-assembled, taxol-stabilized bovine microtubules. Finally, the herbicide 2,6-dichlorobenzonitrile, which inhibits cellulose synthesis in plants, was found to bind specifically to PttMAP20. Together with the known function of cortical microtubules in orienting cellulose microfibrils, these observations suggest that PttMAP20 has a role in cellulose biosynthesis.

2,6-Dichlorobenzonitrile (DCB; [Fig. 1A][1] ) was reported to inhibit cellulose synthesis more than 30 years ago ([Hogetsu et al., 1974][2]) and has subsequently been used in numerous studies (e.g. [Montezinos and Delmer, 1980][3]; [Edelmann and Fry, 1992][4]; [Shedletzky et al., 1992][5]; [Suzuki

In soil the herbicide 2,6-dichlorobenzonitrile (dichlobenil) is degraded to the persistent metabolite 2,6-dichlorobenzamide (BAM) which has been detected in 19% of samples taken from Danish groundwater. We tested if common soil bacteria harbouring nitrile-degrading enzymes, nitrile hydratases or nitrilases, were able to degrade dichlobenil in vitro. We showed that several strains degraded dichlobenil stoichiometrically to BAM in 1.5-6.0 days; formation of the amide intermediate thus showed nitrile hydratase rather than nitrilase activity, which would result in formation of 2,6-dichlorobenzoic acid. The non-halogenated analogue benzonitrile was also degraded, but here the benzamide intermediate accumulated only transiently showing nitrile hydratase followed by amidase activity. We conclude that a potential for dichlobenil degradation to BAM is found commonly in soil bacteria, whereas further degradation of the BAM intermediate could not be demonstrated.

The effects of 2,6-dichlorobenzonitrile (DCB), a cellulose biosynthesis inhibitor, on the density (number/μm2), the terminal complex (TC) length, the number of transverse rows of TC subunits of cellulose synthesizing linear TCs, the cellulose microfibril width and the structure and morphology of cell wall and colonies of Erythrocladia subintegra were investigated. Treatment with 50 μM of DCB caused a partial inhibition of cellulose synthesis, whereas with higher concentrations (75–100 μM) no TCs were observed; this shows that DCB at these concentrations inhibits cellulose synthesis completely. The cellulose synthesis inhibition had a direct impact on the morphology of cells and colonies as well as on the cell wall structure and function. Ultrathin sections of E. subintegra fixed cells, when treated with cellobiohydrolase I (CBH I)–colloidal gold complex (10 nm gold particles), showed significant gold particle labelling on the cellulose microfibrils. In order to better visualize the inhibitory eff...

Pectin, an important cell wall component of dicotyledonous plants, is probably the most complex macromolecule in nature. Here, we critically summarize the large amount of data on pectin structure. An alternative model for the macromolecular structure of pectin is put forward, together with ideas on

Summary There has been an ongoing debate about the presence and function of cellulose in pollen tube walls. In this study, the role of cellulose in pollen tube growth was analyzed by growing pollen tubes in vitro in the presence of 2,6-dichlorobenzonitrile (DCB), a specific inhibitor of cellulose biosynthesis. Pollen tubes of Lilium auratum and Petunia hybrida were cultured in vitro in the presence of 0.25 to 10 μmol/L DCB. Although pollen germination and growth rate were not affected, petunia and lily pollen tubes frequently ruptured or assumed a distorted, bulbous shape in the presence of DCB. When DCB was added to normally growing petunia pollen tubes, pollen tube rupture was observed within 15 min. Immunolocalization studies showed that in lily pollen tubes treated with DCB, callose deposition occurred. Ultrastructural examination of DCB treated lily pollen tubes revealed a highly disorganized pollen tube wall. Our results suggest that the reduction or lack of cellulose in the presence of DCB causes significant distortion and weakening of the pollen tube cell wall.

Cellulose is an important component of cell wall, yet its location and function in pollen tubes remain speculative. In this paper, we studied the role of cellulose synthesis in pollen tube elongation in Pinus bungeana Zucc. by using the specific inhibitor, 2, 6-dichlorobenzonitrile (DCB). In the presence of DCB, the growth rate and morphology of pollen tubes were distinctly changed. The organization of cytoskeleton and vesicle trafficking were also disturbed. Ultrastructure of pollen tubes treated with DCB was characterized by the loose tube wall and damaged organelles. DCB treatment induced distinct changes in tube wall components. Fluorescence labeling results showed that callose, and acidic pectin accumulated in the tip regions, whereas there was less cellulose when treated with DCB. These results were confirmed by FTIR microspectroscopic analysis. In summary, our findings showed that inhibition of cellulose synthesis by DCB affected the organization of cytoskeleton and vesicle trafficking in pollen tubes, and induced changes in the tube wall chemical composition in a dose-dependent manner. These results confirm that cellulose is involved in the establishment of growth direction of pollen tubes, and plays important role in the cell wall construction during pollen tube development despite its lower quantity.

Segments cut from the next-to-last (peduncular-1) internode of Avena sativa L. cv. Victory (oat) shoots elongate as much as 10-fold in response to gibberellic acid (GA 3 ). The objective of the present investigation was to differentiate the effects of GA 3 on growth from its effects on wall synthesis (measured gravimetrically and through the incorporation of [ 14 C]-glucose) by using several cell wall synthesis inhibitors with widely varying mechanisms of action. Four compounds, viz. monensin, cycloheximide, lanthanum, and galactose, caused (1) relatively little inhibition of either cell wall synthesis or elongation in segments without GA 3 , (2) roughly proportionate, dose-dependent inhibition of elongation and wall synthesis in GA 3 -treated segments and (3) generally greater inhibition of GA 3 -promoted uptake of radioactivity than of wall incorporation or elongation. Two other compounds, colchicine and 2,6-dichlorobenzonitrile (DCB), (1) inhibited GA 3 -induced elongation considerably more than cell wall synthesis and (2) caused swelling (radial expansion), especially of GA 3 -treated segments. DCB-treated internodal cells apparently compensated for inhibited cellulose synthesis by greater synthesis of matrix polysaccharide (beginning between 3 and 6 h). While normal cellulose synthesis was not required for short-term (up to 6 h) GA 3 -induced elongation or for long-term hormone-promoted radial expansion, it was required for sustained GA 3 -induced elongation. These results indicate that GA 3 -promoted cell wall loosening (manifested as radial expansion) and cell wall synthesis in Avena internodes occur at least partially independently of any hormonal effect on the orientation of microtubules and microfibrils

SUMMARY Evidence from high-resolution images of primary cell walls suggests that the cell wall is constructed from at least two independent yet coextensive fibrous networks, one based on cellulose/hemicellulose and the other on pectin. The ability to analyse the structure of each of these networks in isolation has been hampered by a lack of suitable biological material such as mutants. However, the recent use of the cellulose-synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) that prevents the formation of the cellulose-xyloglucan network while allowing the pectin network to form a functional wall offers the unique opportunity of studying at least the pectin network independently. A range of electron microscopy techniques and a novel spectroscopy method are used to study the walls from tomato suspension cells adapted to growth on DCB. Measurements of the minimum cell wall thickness derived from thin sections of dehydrated walls show that the marked reduction in level of the cellulose/hemicellulose network affects neither the thickness of the wall formed, nor the apparent spacing of pectin molecules. However, images obtained by the fast-freeze, deep-etch, rotary-shadowed (FDR) replica technique show that the three-dimensional architecture of these pectin-rich walls is very different from that of nonadapted walls. Fourier transform infrared (FTIR) microspectroscopy data and immunogold-labelling studies provide additional evidence that supports the previous biochemical data.

Here, we determined the staining properties of Balamuthia mandrillaris cysts, and assessed the effect of 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, and calcofluor white, a brightening agent, on its encystment. Periodic acid-Schiff reagent stained the inner wall intensely and middle and outer walls weakly suggesting that the cyst wall of B. mandrillaris may contain glycans. Furthermore, cysts, but not trophozoites, fluoresced when stained with calcofluor white. Calcofluor white and DCB, a cellulose synthesis inhibitor, inhibited B. mandrillaris encystment. This is the first report suggesting possible glycan biosynthesis in B. mandrillaris encystment, and this pathwaymay provide a potentially useful drug target and help improve treatment.

The rigid component of the cell walls of red macroalgae, cellulose, is lacking in the red microalgae. Instead, the cells are encapsulated within an amorphous polysaccharide. These complex sul fated polysaccharides are composed of at least 10 different sugars, but their structure is not known, When the herbicide 2,6-dichlorobenzonitrile (DCB), a compound that specifically inhibits cellulose biosynthesis, was applied to cultures of the red microalga Rhodella reticulata upon inoculation, growth was inhibited. When added during the stationary phase of growth (after cell division had ceased), DCB did not affect cell number but it did inhibit polysaccharide production. A spontaneous mutant resistant to DCB was selected; it had physiological characteristics similar to those of the wild-type parent. The composition of the cell wall polysaccharide of the mutant was totally modified, being composed almost entirely (98% of its dry matter, as compared to 2.9% in the wild type) of methyl galactose, but retaining the same sulfate content. The molecular mass of the mutant polysaccharide was, however, similar to that of the wild-type parent (∼6 × 106 daltons), although its viscosity was significantly lower.

N-Methylcycloalkano[b]indoles 1a, 1c–d and N-methylindeno[1,2-b]indole (1e) react with 2,6-dichlorobenzonitrile oxide (2) to afford regioselectively propellanes 3. Catalytic hydrogenation of compounds 3 proceeds via the labile β-hydroxyimines 4, which are further decomposed to the starting indole and 2,6-dichlorobenzonitrile. In one case the intermediate imine 4e is stable and isolated in pure form.

Our previous work (E. Shedletzky, M. Shmuel, D.P. Delmer, D.T.A. Lamport [1990] Plant Physiol 94:980-987) showed that suspension-cultured tomato cells adapted to growth on the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a markedly altered cell wall composition, most notably a markedly reduced level of the cellulose-xyloglucan network. This study compares the adaptation to DCB of two cell lines from dicots (tomato [Lycopersicon esculentum] and tobacco [Nicotiana tabacum]) and a Graminaceous monocot (barley [Hordeum bulbosum] endosperm). The difference in wall structures between the dicots and the monocot is reflected in the very different types of wall modifications induced by growth on DCB. The dicots, having reduced levels of cellulose and xyloglucan, possess walls the major integrity of which is provided by Ca(2+)-bridged pectates because protoplasts can be prepared from these cells simply by treatment with divalent cation chelator and a purified endopolygalacturonase. The tensile strength of these walls is considerably less than walls from nonadapted cells, but wall porosity is not altered. In contrast, walls from adapted barley cells contain very little pectic material and normal to elevated levels of noncellulosic polysaccharides compared with walls from nonadapted cells. Surprisingly, they have tensile strengths higher than their nonadapted counterpart, although cellulose levels are reduced by 70%. Evidence is presented that these walls obtain their additional strength by an altered pattern of cross-linking of polymers involving phenolic components. Such cross-linking may also explain the observation that the porosity of these walls is also considerably reduced. Cells of adapted lines of both the dicots and barley are resistant to plasmolysis, suggesting that they possess very strong connections between the wall and the plasma membrane.

The reactions of 2,6-dichlorobenzonitrile oxide with some cytosine nucleosides afforded diastereomeric mixtures of [3+2] cycloaddition products.

The effects of 2,6-dichlorobenzonitrile (DCB, a known inhibitor of cellulose synthesis) and Tinopal LPW (TPL, an agent which disrupts glucan crystallization) on the structure of cellulose synthesizing complexes (terminal complexes, TCs) in the xanthophycean algaVaucheria hamata were investigated. DCB (10 μM) inhibits nascent fibril formation from the TC subunit (based on the absence of impressions) although it does not alter the overall shape of the rectangular TC during the short treatment of 20 min. With a prolonged treatment (60 min), the arrangement of TC subunits becomes disordered, and particles generally exhibited as doublets of subunits are released from each other. DCB also interferes with the formation of the overall shape of the TC although it does not disturb the conversion into TC rows of the subunits (the zymogenic precursor of the TC) packed in the globules. A 15 min treatment with TPL (1 mM) destroys the TC integrity by reducing the subunits into small fragments or particulate aggregates. The particulate rows of the TC are interrupted at many points, and fragments and particulate aggregates are dispersed by prolonged treatment (45 min) with TPL. Unlike DCB, TPL inhibits the conversion of globule subunits into TC rows. New insights on the structural characteristics necessary for cellulose microfibril assembly and possible mechanisms for the biogenesis of theVaucheria TC come from these data.

N-Methylcycloalkano[b]indoles 1a, 1c–d and N-methylindeno[1,2-b]indole (1e) react with 2,6-dichlorobenzonitrile oxide (2) to afford regioselectively propellanes 3. Catalytic hydrogenation of compounds 3 proceeds via the labile β-hydroxyimines 4, which are further decomposed to the starting indole and 2,6-dichlorobenzonitrile. In one case the intermediate imine 4e is stable and isolated in pure form.

Polysaccharide synthesis inhibitors 2,6-dichlorobenzonitrile (DCB) at 10 −6 M, monensin at 10 −7 and galactose at 3 × 10 −3 M and above suppressed elongation growth of intact rice coleoptiles submerged in water. Auxin (indoleacetic acid)-induced elongation of excised rice coleoptiles was suppressed by monensin and galactose, but not by DCB at these concentrations. Neither monensin nor galactose influenced the minimum stress-relaxation time and the relaxation rate of the cell wall of coleoptile segments. DCB inhibited the incorporation of [ 14 C]glucose into the cellulosic polysaccharides without affecting that into the matrix polysaccharides, while monensin and galactose inhibited the incorporation into both the matrix and the cellulosic polysaccharides. Thus, only the synthesis of matrix polysaccharides is required for auxin-induced elongation and the synthesis is not directly related to cell wall loosening in rice coleoptile segments. The synthesis of matrix polysaccharides may contribute to auxin-induced cell elongation by fixing or extending polymers already present in the cell wall.