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  • 1
    ISSN: 1615-6102
    Keywords: Vesicles ; Phycomyces ; Calcium ionophore ; Chitin synthetase ; Invertase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Hyphal elongation, chitin synthesis in vivo, and invertase secretion inPhycomyces blakesleeanus were all inhibited almost instantly by the addition of 5–10 μM calcium ionophore A 23187. Protein biosynthesis was inhibited in these conditions by 30–50%. The ionophore did not affect cell respiration for at least 40 min. Effect on chitin biosynthesis was not due to alterations of the chitin synthetase levels or its activity; nor to impairement in GlcNAc metabolism. In drug-treated cells the number of apical vesicles was severely reduced even at very short periods of incubation, and these low numbers remained constant for at least 60 min of incubation with the ionophore. We suggest that the ionophore collapses the cellular calcium gradient and/or interferes with the normal electrical transhyphal current. As a consequence, formation and migration of apical vesicles are inhibited. These results are further evidence of the role of vesicles in fungal tip growth and exhibit the fact that active chitin synthetase is short-lived in vivo demanding its continuous supply by chitosomes to the cell surface.
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  • 2
    ISSN: 1617-4623
    Keywords: Keywords Amplified fragment length polymorphism  ; Fungal dimorphism  ;  DNA methylation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A modification of the amplified fragment length polymorphism technique was developed for the determination of DNA methylation in dimorphic fungi representative of three of the major fungal taxa: Mucor rouxii, a zygomycete; Yarrowia lipolytica, an ascomycete; and Ustilago maydis, a basidiomycete. DNA obtained from the yeast or mycelial stages of the fungi was digested with a mixture of EcoRI, and one of the isoschizomers MspI and HpaII, whose ability to cleave at the sequence CpCpGpG is affected by the methylation state. The resulting fragments were ligated to primers and subjected to a double round of amplification by the polymerase chain reaction, radiolabeled in the second round, and separated by polyacrylamide gel electrophoresis. Comparison of patterns revealed differences indicative of fragments whose methylation state did or did not change during the dimorphic transition. These results indicate the usefulness of the method for the study of DNA methylation, demonstrate the universality of DNA methylation in fungi, and confirm that differential DNA methylation occurs during fungal morphogenesis.
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  • 3
    ISSN: 1572-9699
    Keywords: Chitin synthetase ; chitosomes ; Mucorales
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Stability of chitin synthetase in cell-free extracts from mycelial fungi was markedly improved by the presence of sucrose in the homogenization media. Breakage of mycelium in sucrose-containing buffer yielded enzyme preparations from which chitosomal chitin synthetase could be purified by a procedure involving ammonium sulfate precipitation, gel filtration and centrifugation in sucrose density gradients. Purified chitosomes catalyzed the synthesis of chitin microfibrils in vitro upon incubation with substrate and activators. Chitosomal chitin synthetase from the filamentous form of M. rouxii was similar to the enzyme from yeast cells, except for the poorer stability and diminished sensitivity to GlcNAc activation of the former.
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  • 4
    ISSN: 1572-9699
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Escherichia coli was grown under various culture conditions. Variations in the levels of formate dehydrogenase which reacts with methylene blue (MB) or phenazine methosulfate (PMS) (N enzyme), formate dehydrogenase which reacts with benzyl viologen (BV) (H enzyme), formate oxidase and hydrogenlyase were analyzed. It was observed that formate dehydrogenase N and formate oxidase were induced by nitrate and repressed by oxygen. Synthesis of formate dehydrogenase H and hydrogenlyase was induced by formate and repressed by nitrate and oxygen. Selenite was required for the biosynthesis of formate dehydrogenase H and hydrogenlyase. Activity of both formate oxidase and hydrogenlyase was inhibited by azide and KCN but not by N-heptyl hydroxyquinoline-N-oxide (HOQNO); on the other hand, formate oxidase was extremely sensitive to HOQNO. Data were obtained which suggest that cytochromes are not involved in hydrogen formation from formate.
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  • 5
    ISSN: 1572-9699
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cell walls from Morchella sp. were isolated after mechanical breakage of the fungus. Analysis of the material revealed that the most prominent compound was protein, phosphate being present in lesser amounts. Non-nitrogenous polysaccharides accounted for 17%. They were mostly soluble in hot HCl. The following monosaccharides were identified by chromatography of hydrolyzed samples: glucose, mannose and galactose. No lipids were detected in the purified cell walls. Chitin was present in a concentration of 16%. It was identified by cytochemical reactions, infrared spectra, and X-ray diffraction analysis. The cell wall of Morchella did not contain chitosan nor cellulose.
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  • 6
    ISSN: 1572-9699
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Properties of β-glucan synthetase from S. cerevisiae were studied. The enzyme exhibited optimal activity at pH 6.7 and 24 C. Km for UDP-glucose was 0.12mm. Addition of Mg++ or Mn++ stimulated its activity by 60% and 21% respectively. High concentrations of EDTA and hydroxyquinoline were inhibitory. Glucan synthetase was fully active in cell-free extracts. Small concentrations of trypsin or subtilopeptidase A from Bacillus subtilis, caused only a slight increase in glucosyl transferase activity, but larger concentrations destroyed β-glucan synthetase. Acid proteases were neither stimulatory nordestructive. Thus it seemsunlikelythat β-glucan synthetase exists in a zymogen form. Glucan synthetase was unstable. It was inactivated more rapidly at 28 C than at 0 C. The presence of substrate, β-glucan or the protease inhibitors PMSF, Antipain or Pepstatin A did not protect β-glucan synthetase from inactivation. Glucan synthetase was not stimulated by addition of cellobiose or β-glucans. The synthesis of β-glucans was competitively inhibited by UDP (Ki=0.45mm). Glucono-δ-lactone, a known inhibitor of β-glucosidases was a strong non-competitive inhibitor of β-glucan synthetase.
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  • 7
    ISSN: 1572-9699
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cell-free extracts from Aspergillus flavus catalyzed the synthesis of chitin from UDP-GlcNAc. Most of the activity was associated with membrane-rich fractions whereas no activity was detected in the cell walls. Chitin synthetase was activated by fungal acid proteases; animal and plant proteases destroyed it. Upon incubation at 0 C and 28 C chitin synthetase was inactivated, probably by the action of proteases present in the particulate preparations. Maximal activity was obtained at pH 6.6–7.1 and 15 C. Arrhenius plot showed a biphasic curve with the transition at 7 C. E values were 3300 Kcal/mole above this temperature and 15500 Kcal/mole below it. The enzyme was activated by GlcNAc and required a divalent metal, the most active being Mg++. By plotting v vs UDP-GlcNAc concentration a sigmoidal curve was obtained. Km calculated at high substrate concentrations was 20mm. Chitin synthetase was competitively inhibited by polyoxin D (Ki 6.5 μm) and UDP (Ki 1.35mm), the latter giving complex kinetics.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 122 (1984), S. 178-190 
    ISSN: 1615-6102
    Keywords: Chitin synthetase localization ; Chitosome sedimentation ; Fungus ; Miniorganelles ; Mucor rouxii ; Plasma membrane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary This study was undertaken to assess the distribution and localization of chitin synthetase in a fungal cell and to evaluate the sedimentation behavior of chitosomes (microvesicular containers of chitin synthetase). Chitosomes were isolated from cell-free extracts of yeast cells ofMucor rouxii by rate-zonal and isopycnic sedimentation in sucrose density gradients. Because of their small size and low density, chitosomes were effectively separated from other subcellular particles. Rate-zonal sedimentation was a suitable final step for isolating chitosomes as long as ribosomes had been eliminated by enzymic digestion. By isopycnic centrifugation, chitosomes could be separated directly from a crude cell-free extract; they cosedimented with a sharp symmetrical peak of chitin synthetase at a buoyant density of d=1.14–1.15g/cm3; the only significant contaminants were particles of fatty acid synthetase complex. From such sedimentations, we estimated that 80–85% of the chitin synthetase activity in the cell-free extract was associated with chitosomes; the rest was found in two smaller peaks sedimenting at d=1.19–1.20 and d=1.21–1.22 (5–10%), and in the cell wall fraction (5–10%). By consecutive rate-zonal and isopycnic sedimentations, chitosome preparations with relatively few contaminating particles were obtained. Potassium/sodium phosphate buffer (pH 6.5)+MgCl2 was the most effective isolation medium for chitosomes. Other buffers such as TRIS-MES+MgCl2 led to massive aggregation of chitosomes and a change in sedimentation properties. This tendency of chitosomes to aggregate could explain why most of the chitin synthetase activity of a fungus is sometimes found associated with other subcellular structures,e.g., plasma membrane.
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  • 9
    ISSN: 1617-4623
    Keywords: Key wordsUstilago maydis ; Zea mays ; Corn smut ; Meiosis ; Cell cycle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The heterobasidiomycetes responsible for plant smuts obligatorily require their hosts for the completion of the sexual cycle. Accordingly, the sexual cycle of these fungi could so far be studied only by infecting host plants. We have now induced Ustilago maydis, the causative agent of corn smut, to traverse the whole life cycle by growing mixtures of mating-compatible strains of the fungus on a porous membrane placed on top of embryogenic cell cultures of its host Zea mays. Under these conditions, mating, karyogamy and meiosis take place, and the fungus induces differentiation of the plant cells. These results suggest that embryogenic maize cells produce diffusible compounds needed for completion of the sexual cycle of U. maydis, as the plant does for the pathogen during infection.
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  • 10
    ISSN: 1572-9699
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract During a screening for methionine-decomposing bacteria several strains were isolated from soil. One of the most active was studied further. It belongs to the genusAchromobacter. As it could not be identified with any of the known species its morphological and biochemical characteristics are presently given.
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