Intracellular urease activity in the lichen Cladonia verticillaris, and its implication for toxicity.

Urea is currently used as a nitrogen fertilizer in many plant cultures, such as sugar cane. Several lichen species grow in the edges of the fields fertilized with urea. This implies that the hydrolysis of an excess of urea by soil bacteria or by the lichens themselves would increase the concentration of ammonia in the lichen thallus to a level that may be toxic to the photobiont. However, Cladonia verticillaris produces urease through positive feedback by urea supplied from the medium. This urease is partially secreted to the media or retained on the external surface of algal cells, as demonstrated herein by an adequate cytochemical reaction. This implies that ammonia produced by urea hydrolysis will be immediately dissolved in the water filling the intercellular spaces on the thallus. A possible protection mechanism against eventual ammonia toxicity, derived from the results described here, is also discussed.

The cell recognition model in chlorolichens involving a fungal lectin binding to an algal ligand can be extended to cyanolichens.

Leptogium corniculatum, a cyanolichen containing Nostoc as photobiont, produces and secretes arginase to culture medium containing arginine. This secreted arginase was pre-purified by affinity chromatography on beads of activated agarose to which a polygalactosylated urease, purified from Evernia prunastri, was attached. Arginase was eluted from the beads with 50 mm alpha-d-galactose. The eluted arginase binds preferentially to the cell surface of Nostoc isolated from this lichen thallus, although it is also able to bind, to some extent, to the cell surface of the chlorobiont isolated from E. prunastri. Previous studies in chlorolichens have shown that a fungal lectin that develops subsidiary arginase activity can be a factor in recognition of compatible algal cells through binding to a polygalactosylated urease, which acts as a lectin ligand in the algal cell wall. Our experiments demonstrate that this model can now be extended to cyanolichens.

Isolation from Gluconacetobacter diazotrophicus cell walls of specific receptors for sugarcane glycoproteins, which act as recognition factors.

Glycoproteins from sugarcane stalks have been isolated from plants field-grown by size-exclusion chromatography. Some of these glycoproteins, previously labelled with fluorescein isothiocyanate, are able to bind to the cell wall of the sugarcane endophyte, N2-fixing Gluconacetobacter diazotrophicus, and largely removed after washing the bacterial cells with sucrose. This implies that sugarcane glycoproteins use beta-(1-->2)-fructofuranosyl fructose domains in their glycosidic moiety to bind to specific receptors in the bacterial cell walls. These receptors have been isolated by affinity chromatography on a sugarcane glycoprotein-agarose matrix, desorbed with sucrose and characterized by sodium dodecyl sulfate polyacrylamide gel electrophresisand capillary electrophoresis (CE).

Cytochemical location of urease in the cell wall of two different lichen phycobionts.

The enzyme urease has been located in the cell wall of recently isolated phycobionts from Evernia prunastri and Xanthoria parietina lichens. Cytochemical detection is achieved by producing a black, electron-dense precipitate of cobalt sulfide proceeding from CO(2) evolved from urea in the presence of cobalt chloride. Cellular fractionation reveals that about 80% of total urease activity was associated to the cell wall on both phycobionts whereas only 20% was recovered as soluble protein.

Enzymatic production of atranorin: a component of the oak moss absolute by immobilized lichen cells.

Cells of the lichen, Evernia prunastri, immobilized in calcium alginate were able to produce the depside atranorin from acetate. The synthesis of the depside was enhanced by molecular oxygen and NADH. This enhancement suggested the participation of an oxidase and an alcohol dehydrogenase to produce an aldehyde-substituted phenolic acid, hematommic acid, as the most probable precursor of atranorin. The participation of both enzymes was confirmed by loading immobilized cells with sodium azide, an inhibitor of several metallo-oxidases, and pyrazole, an inhibitor of alcohol dehydrogenase, which impeded atranorin production and accumulated beta-methyl orsellinate (after azide loading) or its alcohol derivative (after pirazole treatment).

Improvement of the analysis of dansylated derivatives of polyamines and their conjugates by high-performance liquid chromatography.

The paper described a method for improving the hydrolysis of conjugated polyamines in PH fraction, isolated from the lichen Evernia prunastri, as well as the optimization of dansylation procedure of these polyamines on the basis of the pH value to which derivatization is achieved. Dansylated polyamines have been later separated by high-performance liquid chromatography (HPLC) using a gradient elution. Hydrolysis of conjugates requires acid treatment at room temperature rather than at 110 degrees C, as usually described. Dansylation is improved at high pH values, whereas removal of phenolics (mainly evernic acid), from the conjugates requires low pH values.

Bioskin as an affinity matrix for the separation of glycoproteins.

Bioskin is a natural product produced by a mixed culture of Acetobacter xylinum, Saccharomyces cerevisiae and S. pombe cultured on media containing sucrose. It is of fibrillar nature able to retain some proteins, such as cytochrome c, by adsorption, and mainly composed of glucosamine and N-acetyl-D-glucosamine. This makes it possible that, at an adequate pH value, proteins charged as polyanionic molecules, such as catalase, can be retained by ionic adsorption using the positively charged amino groups of the matrix. In addition, bioskin can also be used as an affinity matrix to retain glycoproteins able to perform specific affinity reactions with the amino sugars of the matrix, such as invertase, fetuin or ovalbumin. Its possible use as a chromatographic support is discussed.

Production of phenolics by immobilized cells of the lichen Pseudevernia furfuracea: the role of epiphytic bacteria.

Immobilized lichen cells from the thalli of the lichen Pseudevernia furfuracea, supplied with acetate as the only source of carbon, continuously produced phenolic substances, atranorin and physodic acid, over 23 days. Epiphytic bacteria associated with the lichen thallus grew actively, probably using both acetate and reduced compounds supplied by lichen cells, since their active growth was avoided by including 10 microM 3,3'-dichlorophenyl-1,1'dimethylurea in the bath solution. Penicillin largely impeded the growth of epiphytic bacteria and decreased phenolic production, which was recovered only at the end of the experimental period, just when the bacteria started a slow, but active growth. We suggest the cooperation of epiphytic bacteria in the biosynthesis of both atranotrin and physodic acid.

Binding of soluble glycoproteins from sugarcane juice to cells of Acetobacter diazotrophicus.

Sugarcane produces two different pools of glycoproteins containing a heterofructan as glycidic moiety, tentatively defined as high-molecular mass (HMMG) and mid-molecular mass (MMMG) glycoproteins. Both kinds of glycoproteins can be recovered in sugarcane juice. Fluorescein-labelled glycoproteins are able to bind to Acetobacter diazotrophicus cells, a natural endophyte of sugarcane. This property implies the aggregation of bacterial cells in liquid culture after addition of HMMG or MMMG. Anionic glycoproteins seem to be responsible for the binding activity whereas cationic fraction is not retained on the surface ofA. diazotrophicus. Bound HMMG is competitively desorbed by sucrose whereas MMMG is desorbed by glucosamine or fructose. On this basis, a hypothesis about the discriminatory ability of sugarcane to choose the compatible endophyte from several possible ones is proposed.

Changes in free and conjugated polyamines during starvation of sugarcane juices as analyzed by high-performance liquid chromatography.

Changes of the polyamines (PAs) titer in high- and mid-molecular-mass carbohydrates (HMMCs and MMMCs, respectively) obtained from sugarcane juices stored for 72 h at pH 5.2 or clarified at pH 8.0 have been studied. Cadaverine (CAD) is the most abundant free (S) PA in the MMMC fraction from juices at pH 5.2, whereas putrescine (PUT) was revealed as the main PA at pH 8.0. A slight increase in the free PUT titer can be noted at pH 5.2 for 72 h of juice starvation. PAs from MMMC were mainly conjugated to acid-insoluble (PH) molecules. Accumulation of PH-PAs with the time of starvation was especially significant for PUT and CAD. However, CAD has also been detected in the acid-soluble (SH) fraction and its concentration increases with the time of starvation at pH 5.2. The accumulation pattern of free and conjugated PAs from HMMCs is similar to that found for MMMCs although some differences can be observed. For instance, the increase in free PUT with the time at pH 8.0 was 2.7-times higher in the HMMC fraction than in the MMMC fraction. Conjugated PAs associated to acid-soluble macromolecules (SH fraction) achieved a level in HMMC fractions higher than that observed in the MMMC fraction. Moreover, the reported increase with time that was observed in PH-CAD from the MMMC fraction was not observed in the HMMC fraction, and, finally, the increase in PH-PUT with the time was lower for the HMMC fraction than for the MMMC fraction.

Separation of polyamines, conjugated to DNA, by reversed-phase high-performance liquid chromatography.

Genomic DNA was isolated from the lichen Evernia prunastri in order to analyze by high-performance liquid chromatography the occurrence of polyamines conjugated to the macromolecule. The acid-insoluble (PH) fraction of this DNA contained mainly conjugated spermidine, although small amounts of free putrescine and spermidine were also present. The PH fraction of DNA also contained conjugated evernic acid, the main phenol produced by this lichen species. Conjugation of polyamines to calf thymus DNA was carried out under in vitro conditions. Conjugation was to spermidine and mainly to spermine and produced DNA compactation. Evernic acid enhanced the action of polyamines in order to produce DNA aggregation.

Free and conjugated polyamines and phenols in raw and alkaline-clarified sugarcane juices.

Sugarcane juice contains a lot of sucrose associated with several monosaccharides, defined as low molecular mass carbohydrates (LMMC), as well as some polysaccharides and glycoproteins, which are defined as mid and high molecular mass carbohydrates (MMMC and HMMC, respectively). These three categories of carbohydrates can be separated by size-exclusion chromatography through Sephadex G-10 and Sephadex G-50 columns, but elution profiles change drastically after juice clarification performed by adjusting the pH value of the juice to 8.0. In addition, polyamines and some phenolics are currently associated with carbohydrate preparations, and the distribution pattern of these conjugates also changes after clarification. Polyamine levels generally decrease after juice clarification. Cadaverine is completely removed from the different carbohydrate preparations, whereas spermidine is the main polyamine occurring in association with sugarcane carbohydrates, as free or acid-soluble form in LMMC preparation or as acid-soluble and -insoluble forms in both MMMC and HMMC preparations. Polyamines, presumably spermidine, conjugate to p-hydroxybenzoic acid in LMMC, mostly to caffeic acid in MMMC, and to syringic acid in HMMC preparations. HMMC-associated polyamines appear in both acid-soluble and -insoluble fractions. Syringic acid also occurs in the LMMC preparation, but juice clarification changes it from acid-soluble to free form, and it coelutes with sucrose.

Effect of polyamines on the separation of ovalbumin glycoforms by capillary electrophoresis.

The successful separation of ovalbumin (M(r) 45,000; pI 4.7) glycoforms by capillary electrophoresis in an uncoated fused-silica capillary with different buffer additives is reported. The optimum conditions for obtaining the resolution of glycoforms were 25 mM borate (pH 9.0) containing 0.87 mM spermidine or 0.14 mM spermine. The effects of different concentrations of putrescine, cadaverine, spermidine, spermine and some monoamines or diamines are compared in terms of selectivity factors of ovalbumin peaks. Addition of sodium dodecyl sulfate at a concentration below the critical micelle concentration increased the resolution between the three main peaks of ovalbumin but did not permit their microheterogeneity to be expressed.

Separation of arginase isoforms by capillary zone electrophoresis and isoelectric focusing in density gradient column.

Four major arginase isoforms, I, II, III and IV, have been detected in Evernia prunastri thallus. They differ in terms of both physical and biochemical properties. The isoelectric point (pI) of these proteins has been determined by both isoelectric focusing in density gradient column and high-performance capillary electrophoresis (HPCE). Isoelectric focusing revealed charge microheterogeneity for isoforms II and IV whereas arginases I and II had the same pI value of 5.8. HPCE separation confirmed this charge microheterogeneity for isoform IV but not for isoform III, and provided evidence of microheterogeneity for isoforms I and II. The effect of various electrolyte buffers and running conditions on the HPCE separation of arginase isoform were investigated. Addition of 0.5 mM spermidine (SPD) to the running buffer reduced the electroosmotic flow (EOF) and permitted discriminating between the native proteins and protein fragments.

Analysis of Usnea fasciata crude extracts with antineoplastic activity.

Different fractions, isolated from the lichen Usnea fasciata, were analyzed by PC, TLC, and RP-HPLC. Analysis of the organic phases, mainly containing phenolics, revealed that usnic acid is the main product from secondary metabolites, whereas the polysaccharides isolichenin and raffinose are the most abundant water-soluble carbohydrates. Fractions containing usnic acid, as well as those containing isolichenin, showed moderate activity against sarcoma 180 and Ehrlich tumor cells. High antitumoral activity, near 90% inhibition, was found associated with the fraction containing raffinose.

Ionic adsorption of catalase on bioskin: kinetic and ultrastructural studies.

Bioskin is a natural polymer produced by Acetobacter xylinum and several yeasts in culture. It contains glucosamine and N-acetyl galactosamine which promote ionic adsorption of catalase at the adequate pH value. High values of ionic strength are required to enzyme desorption. Adsorption of catalase on bioskin fibers has been visualized by scanning electron microscopy associated to a dispersion X-ray analyzer. At low enzyme density, the affinity of the immobilized catalase for hydrogen peroxide was 30% lower than that of the free enzyme. This affinity decreased dramatically at higher density of immobilized enzyme and could not be increased by agitation of the enzyme reaction mixture. Immobilized catalase retains about 70% of its initial activity after 16 d storage, whereas soluble enzyme is completely inactivated after 3 d at room temperature. The haeme group of catalase is not protected after immobilization since it is accessible to both EDTA and phloroglucinol, chelating agents which inactivate catalase by removing the iron atom from the haeme group.

Determination of salicylic acid by HPLC in plasma and saliva from children with juvenile chronic arthritis.

A high performance liquid chromatography (HPLC) method has been developed for measuring salicylic acid in the plasma and saliva of children with juvenile chronic arthritis (JCA). Samples were extracted with diethyl ether and, after drying, redissolved in methanol to be chromatographed. Quantitation of salicylic acid was performed by reverse phase HPLC on a spherisorb ODS-2 column, using methanol: water: acetic acid as mobile phase. Phenolic was monitored by absorbance at 237 nm. Linearity between the amount of mass injected and the response in the detector was determined. This method was applied to compare concentrations of salivary and plasma salicylic acid. The method also permitted the quantitation of salivary salicylate as a non-invasive, indirect method for monitoring the concentration of plasma salicylate in patients with JCA.

Purification and Partial Characterization of a Fructanase which Hydrolyzes Natural Polysaccharides from Sugarcane Juice.

A new sugarcane (Saccharum officinarum L.) fructanase which hydrolyzes both high molecular weight polysaccharides mid R:Fructose(4):Galactitol(5)mid R:(n) (SP) and moderate-sized carbohydrates mid R:Fructose(2):Galactitol(3)3mid R:(n) (MMWC) has been purified from sugarcane juice. The K(m) value has been estimated to be 33.7 micrograms per milliliter and 20 micrograms per milliliter for SP and MMWC, respectively. The optimal pH and temperature values are 6.0 and 30 degrees C, respectively. Purified protein has a pl value of 6.35 and a molecular weight of 13.2 kilodaltons. Fructanase activity appears to be Mn(2+)-dependent.

High performance liquid chromatography of the dansyl derivatives of putrescine, spermidine, and spermine.

A high performance liquid chromatographic (HPLC) method, based on dansylation and fluorescence detection, is described for the estimation of putrescine, spermidine, and spermine in lichen (Evernia prunastri [L.]) samples. Because of the high concentrations of phenols and salts, dansylation was followed by a pre-HPLC purification step. Both flow rate and mobile phase (methanol:water) followed a gradient for optimum resolution on a reverse-phase column. Amounts as small as 0.3 picomole of standard polyamines could be detected. In applying the method to lichens, it was found that 5.45% (w/w) of the exogenous putrescine taken up by the thallus was unbound in the algal partner and that 60% (w/w) was conjugated in the thallus, perhaps to lichen phenolics.

An improved method to isolate lichen algae by gel filtration.

Photobiont cells of the lichen Evernia prunastri have completely been separated from their fungal partner by filtration through a bed of Sepharose 2B. Both mannitol and ribitol have been quantified by gas-liquid chromatography in the different steps of the isolation procedure. Absence of mannitol, which is exclusively produced by the mycobiont, has been used as the best probe to monitor isolation.