Effects of cultivation techniques and media on yields and morphology of the basidiomycete Armillaria mellea

Summary The yield of cell mass and the morphology of Armillaria mellea, strain ATCC 11114, was studied using a variety of cultivation methods: solid media, standing liquid culture, shake flasks, tower reactors and impeller-stirred reactors. Two different media, malt extract broth and a glucose/asparagine/peptone-medium, and the corresponding agar media, were used. Yields were higher in the malt extract media than in the glucose media. Generally the highest yields were obtained on solid media while agitated cultures gave the lowest yields. Morphological characteristics such as pellet formation, adhesion to surfaces and pigment production were significantly affected by culture conditions.     

Nonrandom structural features in the heparin polymer

Computer simulation studies were used to prepare an ensemble of heparin number chains. The polydispersity of these chains was simulated by introducing a specific "fraction of terminators", and it closely resembled the experimentally observed polydispersity of a porcine mucosal, glycosaminoglycan heparin. The same percentage of simulated chains contained antithrombin III (ATIII) binding site sequences as are typically found to contain ATIII binding sites using affinity chromatography. Heparin lyase action was then simulated by using Michaelis-Menten kinetics. In one model, heparin chains were constructed from the random assembly of monosaccharide units using the observed mole percentage of each. After simulated depolymerization, the final oligosaccharides formed were compared to the observed oligosaccharide products. The simulation which assumed a random distribution of monosaccharide units in heparin did not agree with experimental observations. In particular, no ATIII binding site sequences were found in the simulated number chains. The results of this simulation indicate that heparin is not simply a random assembly of monosaccharide units. These results are consistent with the known, ordered biosynthesis of heparin. In a second model, heparin chains were constructed from randomly assembled oligosaccharides at the mole percentage in which each is found in the final product mixture. The action of heparin lyase was then simulated, and the distribution of the oligosaccharide products was measured throughout the simulated time course of the depolymerization reaction. The simulated rate of formation and final concentration of a particular oligosaccharide which contains a portion of heparin's ATIII binding site were similar to those observed experimentally. These results are consistent with the random distribution of ATIII binding sites within glycosaminoglycan heparin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of phosphatidylinositol 4,5-bisphosphate proceeds through an intracellular route in rat hepatocytes

The subcellular distribution in rat hepatocytes of enzymes participating in the entire generation cycle of phosphatidylinositol 4,5-bisphosphate, and phosphorylated intermediates of this pathway, has been examined by Nycodenz gradient centrifugation. Our results indicate that the synthesis of phosphatidylinositol takes place in the endoplasmic reticulum, and that its phosphorylation to phosphatidylinositol 4-phosphate occurs intracellularly in low-density membranes before translocation to the plasma membrane, where it is further phosphorylated to phosphatidylinositol 4,5-bisphosphate. The intracellular formation of PIP implies a vesicular transport to the plasma membrane.     

Homogeneous, structurally defined heparin-oligosaccharides with low anticoagulant activity inhibit the generation of the amplification pathway C3 convertase in vitro

This paper demonstrates that heparin-oligosaccharides with low anticoagulant activity have a high capacity to inhibit activation of the amplification pathway of complement in vitro. We prepared heparin-oligosaccharides by partial depolymerization of heparin using purified flavobacterial heparinase. The resulting oligosaccharide mixture was then fractionated using strong anion exchange-high pressure liquid chromatography to produce individual oligosaccharide components of this mixture, with degree of polymerization ranging from 2 to 16. These heparin-oligosaccharides were examined for both their anticoagulant activity and capacity to inhibit activation of the amplification pathway of complement. Although there was little difference among commercial heparins, a correlation between molecular weight and activity to inhibit convertase generation was clearly established for heparin-oligosaccharides between degree of polymerization 2 through 16. Heparin-oligosaccharides of degree of polymerization 10-16 (Mr 3888-5320) demonstrated up to 54% of heparin's activity on a molar basis (and up to 163% of heparin's activity on a weight basis) in inhibiting the amplification pathway of complement in vitro while showing almost no anticoagulant activity. These studies, for the first time, completely separate heparin's ability to inhibit complement activation from its anticoagulant activity.     

Preparation and structure of heparin lyase-derived heparan sulfate oligosaccharides

Porcine intestinal mucosal heparan sulfate was exhaustivelydepolymerized on a large scale using beparin lyase II (heparinaseII) or heparin lyase III (heparitinase, EC 4.2.2.8 [EC] ). The oligosaccharidemixtures formed with each enzyme were fractionated by low pressuregel permeation chromatography. Size-uniform mixtures of disaccharides,tetrasaccharides, and hexasaccharides were obtained. Each size-fractionatedmixture was then purified on the basis of charge by repetitivesemipreparative strong-anion-exchange high-performance liquidchromatography. This approach has led to the isolation of 13homogenous oligosaccharides. The purity of each oligosaccharidewas demonstrated by the presence of a single peak on analyticalstrong-anion-exchange high-performance liquid chromatographyand reversed polarity capillary electrophoresis. The structuresof these oligosaccharides were established using 500 MHz one-and two-dimensional nuclear magnetic resonance spectroscopy.Three of the thirteen structures that were solved were novelwhile the remaining 10 have been previously described. All ofthe structures obtained using heparin lyase III contained a     

Preparation and structural characterization of large heparin-derived oligosaccharides

Porcine mucosal heparin was partially depolymerized with heparinlyase I and then fractionated into low-molecularweight (<5000)and high-molecular-weight (>5000) oligosaccharides by pressurefiltration. The high-molecular-weight oligosaccharide mixture({small tilde}50 wt% of the starting heparin) also containedintact heparin. This intact polymer complicates oligosacsharidepurification. Thus, the low-molecular-weight fraction was usedto prepare homogeneous oligosaccharides for structural characterization.The low-molecular-weight oligosaccharide mixture was first fractionatedby low pressure gel permeation chromatography into size-uniformmixtures of disaccharides, tetrasaccharides, hexasaccharides,octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharidesand higher oligosaccharides. Each size-fractionated mixturewas then purified on the basis of charge by repetitive semi-preparativestrong-anion-exchange high-performance liquid chromatography.This approach has led to the isolation of 14 homogeneous oligosaccharidesfrom disaccharide to tetradecasaccharide. The purity of theseheparin-derived oligosaccharides was determined by gradientpolyacrylamide gel electrophoresis, analytical strong-anion-exchangehigh-performance liquid chromatography, capillary electrophoresisand one-dimensional nuclear resonance spectroscopy. The structureof these oligosaccharides was established using 600 MHz two-dimensionalnuclear resonance spectroscopy . The spectral methods used includedhomonuclear correlation spectroscopy, nuclear Overhauser effectspectroscopy and heteronuclear multiple quantum coherence spech-clscopy.The ¹H/¹H connectivities of the protons of each sugar residuein an oligosaccharide were established by two-dimensional homonuclearcorrelation spectroscopy, while ¹H/¹³C assignments were madeusing ¹H inverse detection. One- and two-dimensional nuclearresonance spectroscopic analysis of these heparin oligosaccharidesshowed two closely related groups of heparin-oligosaccharidesare afforded by enzymatic depolymerization of heparin. One groupis fully sulphated, having the structures     

Selected ion monitoring for rapid differentiation of mycobacteria isolated from domestic animals

Gas chromatography/mass spectrometry adapted for selected ion monitoring was used to detect C₃₂ mycocerosic acid in short-term incubated cultures of procineand canine strains of mycobacteria. The method can be employed for rapid differentiation of Mycobacterium tuberculosis from M. avium-intracellulare.     

A new method for sequencing linear oligosaccharides on gels using charged, fluorescent conjugates

A new method is described for sequencing linear oligosaccharides on gels using charged, fluorescent conjugates. The reducing ends of various mono-, di-, tri-, and tetra-saccharides were conjugated with monopotassium 7-amino-1,3-naphthalenedisulfonate (a fluorescent and negatively charged compound) by reductive amination using sodium cyanoborohydride. The sugar conjugates were purified by preparative gradient polyacrylamide gel electrophoresis followed by a newly developed technique involving their semi-dry transfer to positively charged nylon membranes and elution with sodium chloride. The structures of a monosaccharide- and trisaccharide-conjugate were established by f.a.b.-m.s. and 2D n.m.r. Seven linear oligosaccharide-fluorescent conjugates were treated sequentially with exoglycosidases and with endoglycosidases. Analysis of the products by gel electrophoresis provided sequence information. These methods may be useful for sequencing oligosaccharides that are chemically or enzymically (endoglycosidase) released from glycoproteins, glycolipids, and proteoglycans.