[Tumour-like lesion of the forefoot due to a retained toothpick fragment. Case report]. / Tumoru podobná afekce prednozí na podklade retence dreveného párátka. Kazuistika.

The case of a 17-year-old patient with a pseudotumour of the forefoot caused by a retained toothpick fragment is reported. The patient had several examinations in a two-year period and was treated for synovialitis of the first and third metatarsophalangeal joints. However, radiography of the plantar surface was the only examination done during these two years. Therapy was unsuccessful. After admission to our department, ultrasonography was performed and a foreign body in granulation tissue was detected. Computed tomography and MRI confirmed the finding. The foreign body granuloma was removed by surgery and the patient healed successfully. Options for visualising wooden foreign bodies not detected on X-ray images are discussed. Key words:pseudotumour, forefoot, toothpick, retained.

Evaluation of the silica microbead method for isolation of red beet protoplast plasma membrane sheets.

The silica microbead procedure was utilized for the isolation of plasma membrane sheets from protoplasts of a higher plant, the red beet (Beta vulgaris L.). Membrane yields, as determined by recovery of an exogenous membrane marker were approx. 75%. The plasma membrane fraction contained the enzyme marker, pH 6.5, vanadate-sensitive, K+-stimulated, Mg2+-ATPase and small amounts of mitochondria, endoplasmic reticulum, and possibly tonoplast. The silica microbead procedure was also used for the isolation of intact vacuoles from microbead-coated protoplasts.

Density gradient localization of plasma membrane and tonoplast from storage tissue of growing and dormant red beet : characterization of proton-transport and ATPase in tonoplast vesicles.

Membranes from homogenates of growing and of dormant storage roots of red beet (Beta vulgaris L.) were centrifuged on linear sucrose gradients. Vanadate-sensitive ATPase activity, a marker for plasma membrane, peaked at 38% to 40% sucrose (1.165-1.175 grams per cubic centimeter) in the case of growing material but moved to as low as 30% sucrose (1.127 grams per cubic centimeter) during dormancy.A band of nitrate-sensitive ATPase was found at sucrose concentrations of 25% to 28% or less (around 1.10 grams per cubic centimeter) for both growing and dormant material. This band showed proton transport into membrane vesicles, as measured by the quenching of fluorescence of acridine orange in the presence of ATP and Mg(2+). The vesicles were collected on a 10/23% sucrose step gradient. The phosphate hydrolyzing activity was Mg dependent, relatively substrate specific for ATP (ATP > GTP > UTP > CTP = 0) and increased up to 4-fold by ionophores. The ATPase activity showed a high but variable pH optimum, was stimulated by Cl(-), but was unaffected by monovalent cations. It was inhibited about 50% by 10 nanomolar mersalyl, 20 micromolar N,N'-dicyclohexylcarbodiimide, 80 micromolar diethylstilbestrol, or 20 millimolar NO(3) (-); but was insensitive to molybdate, vanadate, oligomycin, and azide. Proton transport into vesicles from the 10/23% sucrose interface was stimulated by Cl(-), inhibited by NO(3) (-), and showed a high pH optimum and a substrate specificity similar to the ATPase, including some proton transport driven by GTP and UTP.The low density of the vesicles (1.10 grams per cubic centimeter) plus the properties of H(+) transport and ATPase activity are similar to the reported properties of intact vacuoles of red beet and other materials. We conclude that the low density, H(+)-pumping ATPase of red beets originated from the tonoplast. Tonoplast H(+)-ATPases with similar properties appear to be widely distributed in higher plants and fungi.

Biological Activity of the Isomeric Forms of Helminthosporium sacchari Toxin and of Homologs Produced in Culture.

The effect of Helminthosporium sacchari (HS) toxin isomers and related, pathogen-produced compounds on dark CO(2) fixation in HS-susceptible sugar cane leaf slices was investigated. HS toxin consists of a mixture of three isomeric bis-5-O-(beta-galactofuranosyl)-beta-galactofuranosides (A, B, and C) differing in the position of one double bond in the sesquiterpene aglycone. Maximum inhibition of dark CO(2) fixation in susceptible sugar cane (CP52-68) occurred within 30 to 40 minutes, and amounts necessary to reach 50% inhibition values typically were approximately 1.7 micromolar for natural toxin mixture ( approximately 2:3:5 mixture of isomers A:B:C) and 4, 6, and 0.7 micromolar for isomers A, B, and C, respectively. Other fractions from cultures of the pathogen consist of comparable mixtures of sesquiterpene isomers but have only 1, 2, or 3 galactofuranose units (HS(1), HS(2), HS(3)) or two alpha-glucopyranose units as well as four beta-galactofuranose units (HS(6)). The lower toxin homologs were not toxic to clone CP52-68, but protected sugar cane from the effects of toxin. Minimum ratios of protectant: toxin giving 95% protection were approximately 50:1, 6:1, and 12:1 for HS(1), HS(2), and HS(3), respectively. HS(2) and HS(3) protected when added up to 12 minutes after toxin as well as when added with or before toxin. Some common plant galactopyranosides were not toxic and did not protect at 500:1 molar excess. The sample of HS(6) was toxic at 500 micromolar, and did not protect against HS toxin. With the availability of purified, homogeneous preparations of HS toxin, homologs, and chemically modified or synthetic analogs, the dark CO(2) fixation assay should prove to be a useful tool for understanding the mode of action of HS toxin.

Fast and efficient purification of yeast plasma membranes using cationic silica microbeads.

A fast and efficient procedure for the purification of plasma membranes of Saccharomyces cerevisiae is described. Protoplasts served as starting material. They were coated with cationic silica microbeads. After lysis, the plasma membranes were washed free from debris and cell organelles. This procedure resulted in a high yield (about 85%) of plasma membranes, as judged by measuring vanadate-sensitive ATPase as a plasma membrane marker. The enzyme was enriched 12-fold relative to the homogenate after lysis. Its specific activity was 1.5--2.0 micromol/min per mg protein, the pH optimum was 6.5, and 10 microM vanadate was sufficient to obtain maximum inhibition. Based on the assay of internal markers and electron microscopic studies, we found our preparation essentially free of contamination from other cell organelles.

Reaction pathways of substrate degradation by an acidic endo-1,4-beta-xylanase of Aspergillus niger.

An acidic endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) of Aspergillus niger catalyzes degradation of linear 1,4-beta-xylooligosaccharides by multiple reaction pathways analogous to those catalyzed by lysozyme and alpha-amylases. Quantitative product analysis of enzyme-substrate mixtures using 1-3H-reducing end-labeled xylooligosaccharides and [U-14C]xylotriose led to the following conclusions: (1) bond cleavage frequencies of xylotriose, xylotetraose and xylopentaose are strongly dependent on substrate concentration; (2) at relatively low concentration of the oligosaccharides the enzyme catalyzes transglycosylic reactions leading to products larger than the substrates; (3) xylobiose and to a low extent also xylose, are utilized as glycosyl acceptors in the transfer reactions; (4) the enzyme-glycosyl intermediates effective in the transfer reactions are formed only from the non-reducing part of oligosaccharides, since no evidence was obtained for condensation of two molecules of oligosaccharides; (5) the enzyme does not catalyze degradation of xylobiose and aryl beta-xylosides at an appreciable rate.

Mechanisms of substrate digestion by endo-1,4-beta-xylanase of Cryptococcus albidus. Lysozyme-type pattern of action.

The action pattern and reaction mechanism of the endo-1,4-beta-xylanase of the yeast Cryptococcus albidus were investigated using reducing-end (1-3H)-labelled and uniformly 14C-labelled beta-1,4-xylooligosaccharides up to xylopentaose. The enzyme was found to catalyze degradation of oligosaccharides also by other pathways than a simple hydrolytic cleavage. Bond-cleavage frequency of xylotriose, xylotetraose and xylopentaose were found to be concentration dependent. At high substrate concentration reactions such as xylosyl, xylobiosyl and xylotriosyl transfer occur and result in the formation of products larger than the starting substrate. Xylose and xylobiose to significant extent enter the reaction pathways as glycosyl acceptors. None of the transglycosylic reactions observed with reducing-end-labelled substrates or acceptors were accompanied by a significant label redistribution from the reducing-end unit, suggesting that the enzyme-glycosyl intermediates effective in the transfer reactions can be formed from the non-reducing-end units of oligosaccharides. Evidence for the formation of a termomolecular shifted complex of beta-xylanase with xylotriose has also been obtained. All features of the degradation of oligosaccharides by beta-xylanase are consistent with the lysozyme-type reaction mechanism.

Substrate-binding site of endo-1,4-beta-xylanase of the yeast Cryptococcus albidus.

The substrate-binding site of endo-1,4-beta-xylanase of the yeast Cryptococcus albidus was investigated using, 1,4-beta-xylooligosaccharides (1-3H)-labelled at the reducing end. Evaluation of the affinities of ten imaginary subsites by the method of Suganuma et al. [1978, J. Biochem. (Tokyo) 84, 293--316] pointed out that the substrate-binding site of the enzyme is composed of four subsites and that the catalytic groups are localized in the centre. The imaginary subsites on the left-hand side of the binding site ('non-reducing-end' side) showed little or no affinity to bind xylosyl residues. For the subsites on the right-hand side of the binding site ('reducing-end' side) negative values of affinity were obtained, which means this region of the enzyme is unfavourable for complexing with xylosyl residues. As a consequence of the asymmetric distribution of negative values of affinity around the binding site, the enzyme displays a strong preference for attacking near the reducing end of the substrate. Regardless of the length of [1-3H]xylooligosaccharides, [1-3H]xylobiose was the prevailing reaction product at an early stage of hydrolysis, and frequency distribution of bond cleavage decreased from the second glycosidic bond towards the non-reducing end. Additional information on the substrate-binding site of C. albidus beta-xylanase was obtained by evaluating the efficiency of xylose, xylobiose, methyl beta-D-xyloside and phenyl beta-D-xyloside to serve as glycosyl acceptors in the transglycosylic reactions proceeding at high concentrations of xylotriose.

Complex reaction pathway of aryl beta-xyloside degradation by beta-xylanase of Cryptococcus albidus.

The extracellular endo-1,4-beta-xylanase of the yeast Cryptococcus albidus catalyzes degradation of aryl beta-xylosides by other reactions than simple hydrolytic cleavage. Liberation of phenol or p-nitrophenol from the corresponding beta-xylosides is accompanied by formation of xylose oligosaccharides and only small amounts of xylose. With the aid of phenyl beta-[U-14C]xyloside synthesized from [U-14C]xylose, it was established that the reaction followed a complex pattern with the rate of phenyl beta-xyloside digestion and appearance of various products varying markedly with time. The reaction involves multiple transglycosylic reaction leading first to phenyl glycosides of xylooligosaccharides, which are subsequently hydrolyzed mainly to xylobiose and xylotriose. At concentrations of phenyl beta-xyloside lower than 100 mM the reaction exhibited a significant lag phase, which was followed by period during which the rate of the degradation of the substrate could be determined. The rate showed a strong sigmoidal dependence on phenyl-beta-xyloside concentration. The lag phase could be eliminated and the initial rate accelerated by addition of xylose oligosaccharides, which are hydrolyzed by beta-xylanase. After disappearance of the added oligosaccharides, the reaction transitionally ceased and then resumed again at a rate comparable to the control without added oligosaccharides. It is proposed that beta-xylanase utilizes for degradation of phenyl beta-xyloside two reaction pathways differing in the nature of glycosyl donors.

Inducible beta-xyloside permease as a constituent of the xylan-degrading enzyme system of the yeast Cryptococcus albidus.

The yeast, Cryptococcus albidus, depending on whether it is grown on xylan or glucose, differs remarkably in the ability to take up inducers of extracellular endo-1,4-beta-xylanase synthesis. In washed, glucose-grown cells the initially low ability to take up xylobiose or methyl beta-D-xylopyranoside, increases during incubation with these compounds after a lag-phase shorter than the induction time of the extracellular beta-xylanase. Using of methyl beta-D-[U-14C]xylopyranoside as a very slowly metabolizable inducer of beta-xylanase it has been established that the increase of the rate of xylobiose or methyl xyloside uptake is due to induction of an active transport system for methyl beta-D-xyloside and beta-1,4-xylooligosaccharides. The system is called beta-xyloside permease. The permease activity of induced cells decreases in the absence of beta-xylanase inducers. The induction of permease as well as its inactivation (degradation) can be prevented with cycloheximide, thus both events appear to be dependent on de novo protein synthesis. In analogy with other active transport systems, beta-xyloside permease function can be effectively blocked by inhibitors of energy metabolism in the cells. The demonstrated example of induction of a permease, for inducers and products of hydrolysis of an extracellular polysaccharide hydrolase, points to a new feature of induction of extracellular enzymes in eucaryotic microorganisms.

Xylan-degrading enzymes of the yeast Cryptococcus albidus. Identification and cellular localization.

During growth on wood beta-1,4-xylans the yeast Cryptococcus albidus produced at least two enzymes which convert the polysaccharide to xylose catabolized by the cells. The enzyme almost completely secreted into culture fluid was identified as an endo-1,4-beta-xylanase. The function of the extracellular beta-xylanase is to hydrolyze xylan to oligosaccharides, mainly to xylobiose and xylotriose, which enter the cell where they are split by the second identified enzyme, a cell-bound beta-xylosidae (xylobiase). Aryl beta-xylosidase activity detected in the culture fluid was snown to be due to low affinity of beta-xylanase for p-nitrophenyl beta-D-xylopyranoside. This property of beta-xylanase was preserved after purification of the enzyme by chromatography on DEAE-cellulose, CM-Sephadex and Biogel A 1.5 m or Biogel P 100. Purified beta-xylanase exhibited certain microheterogeneity after polyacrylamide gel electrophoresis. Both extracellular beta-xylanase and intracellular beta-xylosidase were produced in much lower amounts by the cells grown on glucose than by the cells grown on xylan. This suggested that they are not produced constitutively. The investigated strain was not able to grow on cellulose and the crude and purified beta-xylanase were unable to hydrolyze cellulose or its soluble derivatives.

Induction and inducers of endo-1,4-beta-xylanase in the yeast Cryptococcus albidus.

Extracellular endo-1,4-beta-xylanase synthesis in the yeast Cryptococcus albidus is largely inducible. During growth on wood xylans the yeast produces the enzyme in amounts two orders of magnitude greater than on other carbon sources, including xylose. The enzyme can be induced in washed glucose-grown cells by xylan and beta-1,4-xylooligosaccharides. Among the oligosaccharides only xylobiose was not degraded extracellularly, therefore it appears to be the natural inducer of the enzyme. Xylobiose as a metabolisable inducer is effective at low concentrations and constant availability to cells. At high concentration of xylobiose the inductive effect is less pronounced because of catabolic repression by degradation products. Methyl beta-D-xylopyranoside was found to serve as a non-utilizable inducer of beta-xylanase. The enzyme induced by the glycoside appears to be identical with that produced by the cells during growth on xylan.

Chitin structures of the cell walls of synchronously grown virgin cells of Saccharomyces cerevisiae.

The ability of a lytic beta-glucanase of Arthrobacter GJM-1 to dissolve cell walls of Saccharomyces cerevisiae with exception of the chitin-containing fraction was employed for the isolation of chitin-rich residues of the cell walls of synchronously growing populations of virgin cells. Electron microscopical examination of such wall residues isolated from cells at various stages of the budding cycle showed that the first phase of chitin deposition in the wall corresponds to the formation of an annular structure found as a part of the bud scar after cell division. The annular chitin-rich structure could not be isolated at cell cycle stages preceding the bud emergence and at earliest stages of bud development. The observations confirmed that the annular structure (chitin ring) formed during bud growth represents a major part of total chitin present in the bud scar after septum closure.

Growth of Aureobasidium pullulans on waste water hemicelluloses.

Strain Aureobasidium pullulans capable of utilizing hemicelluloses and xylan was cultivated on processed waste dialysis liquor from the production of viscose fibres, containing about 1.5% hemocelluloses. Basic conditions of biomass production were tested on a laboratory scale. The dialysis waste liquor adjusted with mineral acids to pH 4--5 and supplemented with 0.05% yeast autolyzate and 0.2% ammonium sulphate affords protein yields of about 0.8 g/l, corresponding to 4.0--4.5 g dry biomass. Biomass is isolated together with residual water-insoluble hemicelluloses which are not utilized by the microorganism. The total utilization of hemicelluloses attains about 70%.

Metabolism of 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose in yeast and chick-embryo cells.

2-Deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose have been prepared by tritiation of the corresponding unlabeled 2-fluoro sugars. The tritiated 2-fluoro sugars are phosphorylated and activated by UTP and by GTP to yield UDP-2-deoxy-2-fluoro-D-[3H]glucose, UDP-2-deoxy-2-fluoro-D-[3H]mannose, GDP-2-deoxy-2-fluoro-D-[3H]glucose and GDP-2-deoxy-2-fluoro-D-[3H]mannose in both cell types. The nucleotide derivatives could also be labeled in the nucleotide moiety by feeding the cells with [14C]uridine or [14C]guanosine in the presence of unlabeled 2-fluoro sugar. No evidence was obtained for metabolic steps in which the six-carbon chain of 2-fluoro sugars was not preserved. No epimerisation of the label to 2-deoxy-2-fluoro-D-[3H]galactose could be observed by radioactive gas-liquid chromatography of the enzymatic cleavage products of the different 2-fluoro sugar metabolites isolated from either cell type. Yeast and chick embryo cells both incorporate 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose specifically into glycoproteins, although this incorporation is very low when compared to the incorporation of 2-deoxy-D-[3H]glucose.

Xylan-degrading activity in yeasts: growth on xylose, xylan and hemicelluloses.

The ability to grow in liquid media with D-xylose, xylan from decidous trees, and hemicelluloses from conifers was tested in 95 strains of 35 genera of yeasts and yeast-like organisms. Of 54 strains thriving on xylose, only 13 (genera Aureobasidium, Cryptococcus and Trichosporon) utilized xylan and hemicelluloses as growth substrates. The árowth media of these strains were found to contain xylan-degrading enzymes splitting the substrate to xylose and a mixture of xylose oligosaccharides. The ability of these yeasts to utilize the wood components (hitherto unknown in the genus Crytococcus) makes them potential producers of microbial proteins from industrial wood wastes containing xylose oligosaccharides, xylan, and hemicelluloses as the major saccharide components without previous saccharification.

Lysis of intact yeast cells and isolated cell walls by an inducible enzyme system of Arthrobacter GJM-1.

Bacterium Arthrobacter GJM-1 known in the literature as a good producer of alpha-mannanase was found to accumulate in the culture fluid lytic activities against viable yeast cells during growth on isolated cell walls or beta-glucan fractions of yeast. The accumulation of the lytic activities showed an inducible character. The lytic system produced in the medium containing baker's yeast cell walls was capable of complete solubiliaztion of cell wals in vitro. The system lysed viable cells of a number of yeast species and induced their conversion to protoplasts in an osmotically stabilized medium. The lytic system showed different pH and temperature optima when viable cells or isolated cell walls were used as substrates. The pH optimum of the lysis of isolated cell walls was identical with pH optimum of beta-glucanase activities in the crude system. The results pointed out that in the lysis of intact cells, in addition to beta-glucanases, some other factor is involved. Substantial differences in the nature of the outer and the inner surface of cell walls of Saccharomuces cerevisiae were confirmed in this paper based on the different susceptibility to lysis of the cell walls in vivo and in vitro.

Enzymes of the yeast lytic system produced by Arthrobacter GJM-1 bacterium and their role in the lysis of yeast cell walls.

Yeast lytic system produced by Arthrobacter GJM-1 bacterium during growth on baker's yeast cell walls contains a complete set of enzymes which can hydrolyze all structural components of cell walls of Saccharomyces cerevisiae. Chromatographic fractionation of the lytic system showed the presence of two types of endo-beta-1,3-glucanase. Rapid lysis of isolated cell walls of yeast was induced only by endo-beta-1,3-glucanase exhibiting high affinity to insoluble beta-1,3-glucans and releasing laminaripentaose as the main product of hydrolysis of beta-1,3-glucans. This enzyme was able to lyse intact cells of S. cerevisiae only in the presence of an additional factor present in the Arthrobacter GJM-1 lytic system, which was identified as an alkaline protease. This enzyme possesses the lowest molecular weight among other identified enzyme components present in the lytic system. Its role in the solubilization of yeast cell walls from the outer surface by endo-beta-1,3-glucanase could be substituted by preincubation of cells with Pronase or by allowing the glucanase to act on cells in the presence of thiol reagents. The mechanism of lysis of intact cells and isolated cell walls by the enzymes of Arthrobacter GJM-1 is discussed in the light of the present conception of yeast cell wall structure.

Interaction of concanavalin A with external mannan-proteins of Saccharomyces cerevisiae. Glycoprotein nature of beta-glucanases.

beta-Glucanases secreted into culture fluid by protoplasts or intact cells of the yeast Saccharomyces cerevisiae were investigated for the presence of covalently linked carbohydrates. Gel filtration of the enzymes on Biogel A-1.5m showed that endo-beta-1,3-glucanase is a polydisperse enzyme of high-molecular weight which elutes in about the same volume as external yeast invertase. Exo-beta-glucanase was eluted from the gel as a much lighter enzyme. Endo-beta-1,3-glucanase added to a mixture of extracellular mannoproteins was precipitated by concanavalin A to a similar extent to mannan, invertase and acid phosphatase. Under the same conditions exo-beta-glucanase did not interact with the lectin, but was partially precipitated from the solution in the absence of foreign mannan or mannan-proteins. The results show that endo-beta-1,3-glucanase of S. cerevisiae is a mannoprotein of a similar nature to external invertase and acid phosphatase. However, exo-beta-glucanase appears to be a glycoprotein which does not contain the highly branched mannan polymer in its molecule.

Wall mannan of Saccharomyces cerevisiae. Metabolic stability and release into growth medium.

Selective labelling of cell wall mannan with radioactive precursors in growing Saccharomyces cerevisiae showed that this polysaccharide is metabolically stable during exponential growth. Mannan once inserted into the wall is not subject to turnover or release into the growth medium. However, about 10% of the amount of mannan incorporated into the cell wall fraction can be recovered in the non-dialyzable material isolated from the growth medium. Therefore, the mannan escaping from the cell must be either a mannan de novo synthesized, not trapped in the growing wall structure, or a mannan with a non-structural role. Radioactivity was also retained in the wall fraction of cells pre-labelled with [14C] glucose which pointed to metabolic stability of all cell wall polysaccharides in growing S. cerevisiae.