Isoxaben Inhibits the Synthesis of Acid Insoluble Cell Wall Materials In Arabidopsis thaliana

The effect of the herbicide isoxaben on the incorporation of radiolabeled glucose, leucine, uracil, and acetate into acid insoluble cell wall material, protein, nucleic acids, and fatty acids, respectively, was measured. Dichlobenil, cycloheximide, actinomycin D, and cerulenin, inhibitors of the incorporation of these precursors into these macromolecular components, functioned as expected, providing positive controls. The incorporation of radiolabeled glucose into an acid insoluble cell wall fraction was severely inhibited by isoxaben at nanomolar concentrations. Amitrole, fluridone, ethalfluralin, and chlorsulfuron, as well as cycloheximide, actinomycin D, and cerulenin did not inhibit incorporation of glucose into this fraction, ruling out a general nonspecific effect of herbicides on glucose incorporation. The evidence thus suggests that isoxaben is an extremely powerful and specific inhibitor of cell wall biosynthesis.     

Dispersed lignin in tracheary elements treated with cellulose synthesis inhibitors provides evidence that molecules of the secondary cell wall mediate wall patterning

Mesophyll cells of Zinnia elegans var. Envy that had been induced to differentiate into tracheary elements (TEs) in suspension culture were treated with the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB). The deposition of cellulose into the patterned secondary cell wall thickenings typical of TEs was inhibited as demonstrated by reduced incorporation of [¹⁴C]glucose into acetic/nitric insoluble material and absence of cellulose detectable by fluorescence after staining with Tinopal LPW, polarization optics, or labeling with a specific cellulase. Respiration as indicated by release of ¹⁴CO₂ was inhibited to a much lesser extent, supporting a selective mechanism of action of DCB on the cellulose biosynthetic pathway. Patterned secondary cell wall thickenings were deposited in DCB-treated TEs, but these were smaller and less regularly shaped than those of control TEs. These cellulose-depleted thickenings lacked another abundant component of normal thickenings, the hemicellulose xylan, as indicated by absence of labeling with a specific xylanase or an antibody to xylan. DCB-treated TEs also showed dispersed lignin after staining with phloroglucinol, whereas control TEs contained lignin specifically localized to the secondary cell wall thickenings. Isoxaben, another recently described inhibitor of synthesis of acetic/nitric insoluble cell wall material (putatively cellulose), caused the same absence of detectable cellulose and xylan in the thickenings and dispersed lignin. These data suggest that: (i) the localization of lignin is ultimately dependent on the localization of cellulose; (ii) normal patterned wall assembly in TEs occurs in a self-perpetuating cascade in which some molecules of the secondary cell wall mediate patterning of others.     

Penicillin-induced changes in the cell wall composition of Staphylococcus aureus before the onset of bacteriolysis

To analyze if chemical cell wall alterations contribute to penicillin-induced bacteriolysis, changes in the amount, stability, and chemical composition of staphylococcal cell walls were investigated. All analyses were performed before onset of bacteriolysis i.e. during the first 60 min following addition of different penicillin G doses. Only a slight reduction of the amount of cell wall material incorporated after penicillin addition at the optimal lytic concentration was observed as compared to control cells. However, the presence of higher penicillin G concentrations reduced the incorporation of wall material progressively without bacteriolysis. Losses of wall material during isolation of dodecylsulfate insoluble cell walls were monitored to assess the stability of the wall material following penicillin addition. Wall material grown at the lytic penicillin concentration was least stable but about 30% of the newly incorporated wall material withstood even the harsh conditions of mechanical breakage and dodecylsulfate treatment. Dodecylsulfate insoluble cell walls were used for chemical analyses. While peptidoglycan chain length was unaffected in the presence of penicillin, other wall parameters were considerably altered: peptide cross-linking was reduced in the wall material synthesized after addition of penicillin; reductions from approx. 85% in controls to about 60% were similar for lytic and also for very high penicillin concentrations leading to nonlytic death. O-acetylation was also reduced after treatment with penicillin; this effect paralleled the occurence of subsequent bacteriolysis at different drug concentrations. The results are not consistent with hypotheses explaining penicillin-induced lysis as a result of an overall weakened cell wall structure or an overall activation of autolytic wall enzymes but not conflicting with the model that ascribes penicillin-induced bacteriolysis as the result of a very restricted, local perforation of the peripheral cell wall (murosome-induced bacteriolysis).Abbreviations CL Cross-linking - DNFB 2,4-dinitro-1-fluorobenzole - MIC Minimal inhibitory concentration - OD Optical density at 578 nm - PEN Penicillin G     

Correlation between anatomical characteristics of ethanol organosolv pretreated Buddleja davidii and its enzymatic conversion to glucose

Buddleja davidii is a unique biomass that has many attractive agroenergy features, especially its wide range of growth habitat. The anatomical characteristics of B. davidii were investigated before and after ethanol organosolv pretreatment (one of the leading pretreatment technologies) in order to further understand the alterations that occur to the cellular structure of the biomass which can then be correlated with its enzymatic digestibility. Results showed that the ethanol organosolv pretreatment of B. davidii selectively removes lignin from the middle lamella (ML), which does not significantly disrupt the crystalline structure of cellulose. The removal of ML lignin is a major factor in enhancing enzymatic cellulose‐to‐glucose hydrolysis. The pretreatment also causes cell deformation, resulting in cracks and breaks in the cell wall. These observations, together with characterization analysis of the cell wall polymer material, lend support to the hypothesis that the physical distribution of lignin in the biomass matrix is an important structural feature affecting biomass enzymatic digestibility. Biotechnol. Bioeng. 2010;107: 795–801. © 2010 Wiley Periodicals, Inc.     

Metabolism of 2,4-dichlorophenoxyacetic acid in cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.)

Cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.) incorporated 2,4-dichlorophenoxyacetic acid (2,4-D) into a metabolite fraction which was insoluble in ethanol, water, and hot sodium dodecylsulphate. Further treatment with hot dimethylformamide solubilized a material which by the following criteria appeared to consist of 2,4-D derivatives covalently bound to lignin: i) co-chromatography of radioactivity and of UV-absorbing material upon gel permeation chromatography; ii) spectral similarity with authentic lignins (IR- and UV-spectra, phloroglucinol reaction), 2,4-D appeared to be incorporated as the intact molecule, as shown by comparison of ring- and sidechain-labeled 2,4-D and by detection of monohydroxylated and intact 2,4-D as the major radioactive products of acid hydrolysis. The same compounds were released from the metabolite material which could not be solubilized in dimethylformamide. The incorporation of xenobiotics or their metabolites into lignin, followed by deposition in the cell wall, is suggested as a general pathway for local excretion and detoxification by plant cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4-OH-2,5-D 4-hydroxy-2,5-dichlorophenoxyacetic acid - SDS sodium dodecylsulphate - DMF dimethylformamide     

Cell wall formation in yeast

Summary Incorporation of tritiated glucose into cell walls of growingSaccharomyces cerevisiac andSchizosaccharomyces pombe was studied using electron microscopic autoradiography. The pattern and the extent of labelling ofS. cerevisiae cell walls depended on the cell stage in the cell cycle. Quantitative evaluation of autoradiographs showed that the highest rate of wall synthesis took place during bud growth. The incorporation of new material into the wall of growing bud showed an increasing rate with the magnitude of the bud. The incorporation into the mother cell wall was almost negligible during bud growth. The rate of wall synthesis in double cells decreased during cell division. This period and that before new bud initiation was found to be the time of substantially reduced rate of wall replication inS. cerevisiae. A significant random incorporation was observed into the walls of post-division adult cells, both parental and daughter. The cell walls ofS. pombe were labelled almost exclusively at growing tips. The incorporation of tritiated carbohydrates into non-extensile regions ofS. pombe cell walls was found to be only about 5% of the total wall labelling.     

Chemical Composition of Giant Cells Induced by Tunicamycin and Normal Mycelia of Penicillium citrinum

Growth of Penicillium citrinum was reduced in the presence of tunicamycin. Under this condition, reduction of yield of cell wall was greater than that of cellular protein.

Chitin content in the cell wall was several times higher in giant cells formed from conidia in the presence of tunicamycin than in normal mycelia, while reducing sugar content, presumably reflecting glucan content, did not significantly differ. Galactosamine, which was present in normal mycelia and absent in conidia, could not be detected in giant cells. The amino acid composition of the cell wall and whole cells of giant cells differed distinctly from that of normal mycelia.

Tunicamycin did not significantly inhibit the synthesis of DNA, RNA and protein as judged by incorporation of radioactive precursors, while cell wall synthesis, as judged by incorporation of radioactive N-acetylglucosamine, glucose and alanine into acid insoluble fraction, was inhibited by more than 40% in the presence of 10 μg/ml of tunicamycin. In fungi tunicamycin probably acts primarily as an inhibitor of cell wall glycoprotein synthesis and not of chitin synthesis.

Cyclic nucleotides level also differed distinctly between giant cells and mycelia.     

The initiation of cell wall synthesis in parasynchronous cultures of Schizosaccharomyces pombe

Summary Schizosaccharomyces pombe has been grown in parasynchronous culture to study the synthesis of cell wall material. After a lag period of 2.5h following inoculation the cells began to grow, as measured by optical density, dry weight and cell size. The cell number remained constant until 4.5h after inoculation when approximately 70% of the population divided synchronously. Immunofluorescence studies of the growing cells have shown that new wall material is inserted at the cell apices from 2.5 h after inoculation; this result is supported by radio-isotope labelling data which indicated that synthesis of new cell wall material also commenced 2.5 h after inoculation. The incorporation experiments also demonstrated an interruption in cell wall synthesis during the cell separation stage. The composition of the cell wall material varied during the growth cycle, with maximum nitrogen levels at inoculation and following cell division. No serological differences could be detected in the cell walls during the growth cycle.     

Release of thioredoxin from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with environmental stimuli: solubilization of thioredoxin with ethanol

Thioredoxin is crucial for the maintenance of the redox status of cells of all types. Mammalian thioredoxin is secreted from various types of cells, although the mechanism underlying has not yet been clarified. Previously, we demonstrated that thioredoxin was released from Saccharomyces cerevisiae after treatment with ethanol. In this paper, we show that as well as ethanol, low-pH shock and hypoosmotic shock release thioredoxin. Low-molecular-weight proteins in yeast cells were preferentially released by treatment with ethanol and low-pH shock. A cell wall integrity pathway seems partially involved in the hypoosmotic shock-induced release of thioredoxin. Considerable amounts of thioredoxin were present in the insoluble fractions of the cells, a portion of which was associated with lipid microdomains that are resistant to nonionic detergent at 4°C. The intracellular localization of thioredoxin may influence the efficiency of its release from yeast cells with ethanol.     

Experimental alcoholism in rats. Effect of acute ethanol intoxication on the in vitro incorporation of ( 3 H)leucine into neuronal and glial proteins

Abstract— Ethanol administered in vivo or in vitro during incubation of brain slices was studied with respect to its effect on brain protein synthesis. In the in vivo series the rats were given a single intraperitoneal injection of ethanol 3 h before death. Slices of cerebral cortex and liver were incubated in isotonic saline media containing [³H]leucine. Amounts of free and protein-bound radioactivity were determined. Subcellular fractions and fractions enriched in neuronal perikarya and in glial cells were prepared from cortical slices subsequent to incubation, and the specific radioactivity determined for each cell type. The incorporation of [³H]leucine into brain proteins was inhibited while incorporation into liver proteins was stimulated in ethanol-treated rats. The levels of TCA-soluble radio-activity, however, did not differ between the ethanol group and the controls. In the fractionated material from cerebral cortex, the specific radioactivity in the neuronal fraction was unaffected by ethanol, while the radioactivity in the glial fraction was significantly depressed. In vitro administration of ethanol induced a non-linear response in both brain and liver, with depression of leucine incorporation into proteins of cerebral cortex at all concentrations used. When brain slices were exposed to ethanol in vitro, in concentrations corresponding to the in vivo experiments, a similar reduction of the leucine incorporation into the glial fraction was obtained. Incorporation of leucine into subcellular fractions from whole brain cortex was also investigated. The specific sensitivity of the glial fraction to ethanol is discussed in relation to the involvement of the different cell types with transport processes in the brain.     

Regulation of cell wall synthesis in Avena stem segments by gibberellic Acid

Gibberellic acid induces (a) increased elongation of Avena sativa stem segments, (b) increased formation of cell wall material, measured on the basis of dry weight, and (c) increased incorporation of ¹⁴C-glucose into all fractions of the cell wall material. This increased incorporation of radioactivity correlates well with increased formation of cell wall material and shows a time-course pattern similar to the time course of the elongation response. Approximately one hour after the application of gibberellic acid, the rates both of growth and of incorporation of radioactivity accelerate to about 2-fold over the control rate. Gibberellic acid does not stimulate the incorporation of labeled glucose into the cell wall material simply by increasing the rate of uptake of glucose by internodal cells. The stimulation of the incorporation of ¹⁴C-glucose into cell wall material, which reflects the stimulation of cell wall synthesis, seems to be an important and relatively early effect of gibberellic acid in this system and probably contributes significantly to the elongation response elicited by the hormone.     

Regenerated cell wall of carrot protoplasts isolated from suspension-cultured cells

Protoplasts of Daucus carota L. cultured in a synthetic liquid medium resumed cell division after about 4 days of cultivation. During this lag period, nucleic acid and protein showed only slight increases but the protoplasts commenced cell-wall regeneration soon after the removal of lytic enzymes. The originally spherical protoplasts became ellipsoidal before they underwent division. Radioactive glucose and myo-inositol were readily utilized by the protoplasts. Most of the radioactivity, however, appeared in extracellular polysaccharides and only a small portion was deposited in the regenerated wall. The sugar composition of new cell wall, as studies by chemical analysis and incorporation of labelled precursors, was shown to be considerably different from that of normal cell wall.     

A novel, repeated fed-batch, ethanol production system with extremely long term stability achieved by fully recycling fermented supernatants

Using Saccharomyces cerevisiae, a novel, repeated fed-batch ethanol production system from corn flour by fully recycling fermented supernatants is demonstrated. With recovery of ethanol by evaporation coupled with consecutive removal of the insoluble and soluble inhibitory substances accumulated, either completely or partially by filtration, the concentrations of the soluble inhibitors in the system could be maintained at their equilibria. As a result, a sustained high concentration of ethanol (up to 15% v/v) and significant pollution control performance were obtained.     

Crystallinity of lyophilised carrot cell wall components

The aim of this work was to investigate the effect of removal of cell wall components on the crystallinity of cell walls using X-ray diffraction. Various insoluble cell wall residues were prepared following a sequential extraction of carrot cell wall material. X-ray diffraction patterns were typical of cellulose although there was a possible contribution of pectic polysaccharides to the crystallinity. As more amorphous material was removed to produce a cellulose rich residue, the crystallinity index increased from 12 to 16%, larger than that estimated from cellulose alone. For the last residue treated with 4M KOH, a lower value of crystallinity was found (14%) which resulted from the change of some crystalline domains of cellulose into amorphous regions. Pressing conditions (temperature, water content) have been investigated and did not alter the crystallinity index significantly.     

Physiological and 15N-NMR analysis of molecular nitrogen fixation by Methanococcus thermolithotrophicus, Methanobacterium bryantii and Methanospirillum hungatei

Two mesophilic methanogenic bacteria, Methanobacterium bryantii strain MOH and Methanospirillum hungatei strain GP1 were demonstrated, using several different experimental approaches, to fix dinitrogen. Evidence includes (1) growth with N2 as the sole nitrogen source; (2) incorporation of 15N2 into cellular material (both soluble amino acid pools and insoluble cell protein and other macromolecules) detected by 15N-NMR spectroscopy; (3) acetylene reduction to ethylene by the cells, and inhibition of this reaction by bromoethanesulfonic acid (BES), a methanogen inhibitor. High-resolution 15N-NMR analysis of ethanol extracts of these organisms and cross-polarization magic-angle sample spinning analysis of the solid debris from these extracts are compared to labeled material from Methanococcus thermolithotrophicus, a methanogen previously determined to fix dinitrogen.     

An electron microscopy study of wall expansion during Candida albicans yeast and mycelial growth using concanavalin A-ferritin labelling of mannoproteins

Depending upon growth temperature, Candida albicans can exhibit two different morphologies, a budding yeast or a mycelium. By studying the distribution of concanavalin A-ferritin particles on the cell wall surface during bud and germ tube formation, we have elucidated the way cell wall extension occurs. Both processes initially require the localized lysis of the wall in order to allow the incorporation of the newly synthesized material. Later on, the cell wall behaves as an elastic structure, allowing extension by an intosusception process and, as a consequence, cell growth.Abbreviation Con A concanavalin A     

Electron-energy-loss spectroscopic imaging of calcium and nitrogen in the cell walls of apple fruits

Changes in texture are an integral part of ripening in most fleshy fruits and these changes are thought to be determined, primarily, by alterations in cell wall structure. Electron energy loss spectroscopy (EELS) imaging was used to obtain quantitative information on the levels of calcium and nitrogen in the cell walls of apple (Malus domestica Borkh. cv. Cox's Orange Pippin) fruits. Samples of fruit cortex were prepared for EELS by high-pressure freezing and molecular distillation drying to minimize loss and redistribution of soluble cell wall components such as calcium. The EELS imaging successfully resolved calcium and nitrogen levels in the middle lamella and primary cell wall. When the elemental compositions of the cell walls of Cox's apples from two sites in the UK were compared at harvest or after 6 months storage, the orchard which always produced consistently firmer fruit had significantly lower levels of cell wall calcium and higher levels of cell wall nitrogen. This result was unexpected since firm texture in apples and other fruits has been commonly associated with elevated levels of fruit calcium. The nitrogen-rich material in the sections used for EELS was insoluble in acidified methanol, indicating that it represented a high-molecular-weight component in the cell wall. Furthermore, total tissue hydroxyproline levels were greatest in material with elevated cell wall nitrogen, suggesting enhanced levels of wall structural proteins in the tissue. These data indicate a correlation between increased amounts of cell wall nitrogen and firm fruit texture. The possible role of cell wall proteins in determining the textural properties of fruit tissue is discussed. Received: 19 November 1998 / Accepted: 28 January 1999     

Magnesium deprivation reproduces the coordinate effects of serum removal or cortisol addition on transport and metabolism in chick embryo fibroblasts.

A variety of unrelated effectors stimulate or inhibit coordinately the same array of metabolic reactions in chick embryo fibroblasts, including the uptake of 2-deoxy-D-glucose and uridine, and the incorporation of uridine and thymidine into acid insoluble material. The coordinate inhibition of these reactions by omission of serum or addition of cortisol is reproduced quantitatively by lowering the concentration of magnesium (Mg2+) in medium containing 0.2 mM Ca2+. The response times for the utilization of uridine and thymidine following the removal of addition of Mg2+ are similar to those which follow removal or addition of serum. The effect of serum on the incorporation of choline, which is not part of the coordinate response to unrelated effectors, is not reproduced by varying Mg2+ concentrations. The results support the hypothesis that the availability of Mg2+ within the cell plays a central role in the coordinate control of transport, metabolism and growth by external physiological effectors.     

Effect of Ethylenediaminetetraacetic Acid, Triton X-100, and Lysozyme on the Morphology and Chemical Composition of Isolated Cell Walls of Escherichia coli

Extraction of a partially purified preparation of cell walls from Escherichia coli with the nonionic detergent Triton X-100 removed all cytoplasmic membrane contamination but did not affect the normal morphology of the cell wall. This Triton-treated preparation, termed the “Triton-insoluble cell wall,” contained all of the protein of the cell wall but only about half of the lipopolysaccharide and one-third of the phospholipid of the cell wall. This Triton-insoluble cell wall preparation was used as a starting material in an investigation of several further treatments. Reextraction of the Triton-insoluble cell wall with either Triton X-100 or ethylenediaminetetraacetic acid (EDTA) caused no further solubilization of protein. However, when the Triton-insoluble cell wall was extracted with a combination of Triton X-100 and EDTA, about half of the protein and all of the remaining lipopolysaccharide and phospholipid were solubilized. The material which remained insoluble after this combined Triton and EDTA extraction still retained some of the morphological features of the intact cell wall. Treatment of the Triton-insoluble cell wall with lysozyme resulted in a destruction of the peptidoglycan layer as seen in the electron microscope and in a release of diaminopimelic acid from the cell wall but did not solubilize any cell wall protein. Extraction of this lysozyme-treated preparation with a combination of Triton X-100 and EDTA again solubilized about half of the cell wall protein but resulted in a drastic change in the morphology of the Triton-EDTA-insoluble material. After this treatment, the insoluble material formed lamellar structures. These results are interpreted in terms of the types of noncovalent bonds involved in maintaining the organized structure of the cell wall and suggest that the main forces involved are hydrophobic protein-protein interactions between the cell wall proteins and to a lesser degree a stabilization of protein-protein and protein-lipopolysaccharide interactions by divalent cations. A model for the structure of the E. coli cell wall is presented.