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41.
Summary The ultrastructure and composition of the extracellular matrices (ECMs) associated with germ tubes and appressoria ofColletotrichum lindemuthianum have been examined. Flexuous fibres (fimbriae), up to 6 m long and 4–30 nm in diameter, protruded from the surface of germ tubes and appressoria. Anionic colloidal gold and lectin cytochemistry showed that ECMs of germ tubes and appressoria contain basic proteins, -D-mannose and -D-galactose residues. A monoclonal antibody, UB26, was raised to infection structures isolated from leaves ofPhaseolus vulgaris infected withC. lindemuthianum. UB26 recognised a protein epitope on two glycoproteins (Mr 133,000 and 146,000). Reductions in the Mr of these proteins after treatment with peptide-N-glycosidase and trifluoromethane sulphonic acid suggest that they carry N- and O-linked side-chains. Immunofluorescence and EM-immunogold labelling showed that glycoproteins recognised by UB26 were restricted to the ECMs around germ tubes and appressoria but fimbriae were not labelled. Unlike appressorial germ tubes formed in vitro, intracellular infection hyphae were not labelled, suggesting that the glycoproteins recognised by UB26 are not present on fungal structures formed within host cells. In liquid culture, these glycoproteins were not released into the medium, suggesting they are physically linked to the cell wall. Also, the glycoproteins were not removed from glass surfaces by ultrasonication. These results suggest that glycoproteins recognised by UB26 may be involved in the adhesion of germ tubes and appressoria to substrata. Our results show that the ECMs of germ tubes and appressoria differ markedly in structure and composition from those of conidia and intracellular hyphae, and that extracellular glycoproteins are associated with specific regions of the fungal cell surface.Abbreviations ECM
extracellular matrix
- BPA
Bauhinia purpurea agglutinin
- BSA
bovine serum albumin
- DIC
differential interference contrast
- FITC
fluorescein isothiocyanate
- GNL
Galanthus nivalis lectin
- GSI-B4
Griffonia simplicifolia isolectin B4
- HEPES
(N-(2-hydroxyethyl)piperazine-N-(2-ethanesulphonic acid)
- IIF
indirect immunofluorescence
- IPC
isopycnic centrifugation
- MAb
monoclonal antibody
- PEG
polyethylene glycol
- PBS
phosphate buffered saline
- PNGase
peptideN-glycosidase
- SDS-PAGE
sodium dodecyl sulphate polyacrylamide gel electrophoresis
- TCS
tissue culture supernatant
- TEM
transmission electron microscopy
- TFMS
trifluoromethane sulphonic acid 相似文献
42.
Ingrid Ahrenholtz Michael G. Lorenz Wilfried Wackernagel 《Archives of microbiology》1994,161(2):176-183
A quantitative endonuclease assay, which relies on the introduction of single and double strand breaks into supercoiled plasmid DNA, was used to study the activity of the extracellular nuclease of Serratia marcescens SM6 in buffer and in groundwater. The parallel enzyme concentration-dependent production of relaxed and linear plasmid molecules suggests that the nuclease produces single and double strand breaks in duplex DNA. Bovine serum albumin stimulated the nuclease activity towards DNA and RNA and increased the stability of the enzyme against thermal inactivation. The DNase activity at 4 °C and 50 °C was almost half of that at the optimum temperature (37 °C). The nuclease was active in groundwater, although the specific activity was lower than in buffer. In a groundwater aquifer microcosm, mineral-adsorbed transforming DNA was substantially less accessible to the nuclease than was dissolved DNA. The data suggest that the extracellular nuclease of Serratia marcescens may contribute to DNA turnover in the environment and that adsorption of DNA to minerals provides protection against the nuclease.Abbreviations GW groundwater
GWA groundwater aquifer 相似文献
43.
44.
Martha J. Powell 《Protoplasma》1994,181(1-4):123-141
Summary In development of the primitive fungi, chytridiomycetes, unwalled zoospores bearing single, posterior flagella are transformed into walled, round-cells which elaborate the thallus. Production, structural modification, or release of extracellular material are involved with each transition of developmental stage. This article reviews the variety and developmental changes of extracellular materials found at the cell surface of chytridiomycetes. A cell coat, produced from Golgi-derived vesicles during zoosporogenesis, is visible around free swimming zoospores of some chytridiomycetes. How the zoospore surface receives and transduces signals is not widely explored, but it is known that fenestrated cisternae and simple cisternae, which are integrated into the microbody-lipid globule complex, are spatially and structurally associated with the plasma membrane and flagellar apparatus. This spatial association, as well as the cytochemical localization of calcium in fenestrated cisternae, suggest a mechanism for signal transduction and for regulation of zoospore motility. Zoospores become encased in a new layer of extracellular material as the zoospore encysts. Among some chytrids the source of this material is preexisting vesicles which fuse with the plasma membrane. Among other zoospores, a readily identifiable population of encystment vesicles is not apparent, demonstrating that there is no single pattern or mechanism for zoospore encystment in chytridiomycetes. Encysted zoospores developing into thalli, typically produce cell walls with a microfibrillar substructure. Ultrastructural analysis of walls reveals distinctive architecture and remarkable sculpturing which have been used in systematics of some members of chytridiomycetes. Nothing is known as to underlying controls of cytoskeletal elements and plasma membrane enzyme complexes in wall biogenesis. Many changes in cell surface structures accompany thallus maturation. Septa, many traversed with plasmodesmata, are produced in most chytrid thallus types. As sporangia and resting spores prepare for the production and release of zoospores, additional extracellular layers of material are frequently produced. Polarized deposits of extracellular material become discharge plugs, discharge vesicles, or endoopercula. Interstitial material is also released into cleavage furrows. Circumscissile or localized digestion of walls produce operculate or inoperculate exit ports for zoospore release. Cryofixation preserves more extensive extracellular material than does conventional chemical fixation, and broader application of cryofixation may radically alter our current view of cell surface structure. Thus chytridiomycetes exhibit a range in patterns for the occurrence and subsequent modifications of extracellular materials, even for members within the same order. The most universally recognized role for these extracellular materials is protection. Although there is a reasonable view of the types of extracellular material involved in chytridiomycete development, we have only limited understandings of their biogenesis or roles in regulation and communication, areas awaiting more investigations.Abbreviations DIC
Nomarski-differential contrast optics
- TEM
transmission electron microscopy 相似文献
45.
The Regional Distribution of N-Acetylaspartylglutamate (NAAG) and Peptidase Activity Against NAAG in the Rat Nervous System 总被引:4,自引:4,他引:0
Stefanie Fuhrman Miklos Palkovits Martha Cassidy Joseph H. Neale 《Journal of neurochemistry》1994,62(1):275-281
Abstract: N -Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate and N -acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two-stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate and N -acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was >0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus. 相似文献
46.
Ecto-Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor 总被引:1,自引:0,他引:1
Zofia Pawlowska Michael V. Hogan Elizabeth Kornecki Yigal H. Ehrlich 《Journal of neurochemistry》1993,60(2):678-686
Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ-32P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with 32Pi. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled Pi. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-α-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg2+, implicating this activity in the previously demonstrated regulation of Ca2+-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10–100 μM) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis. 相似文献
47.
Lina Assad Melvin M. Schwartz Ismo Virtanen Victor E. Gould 《Virchows Archiv. B, Cell pathology including molecular pathology》1993,63(1):307-316
Frozen samples of minimal change glomerulopathy (MCG), and of membranous, segmental and diffuse lupus glomerulonephritis (MGN,
SGN, DLGN) were studied to assess the distribution of tenascin (Ten), and the extradomains A and B (EDA-and EDB-) and oncofetal
(Onc-) isoforms of cellular fibronectin (cFn). Cryosections were immunostained by the ABC method with specific monoclonal
antibodies. In MCG, mesangial Ten and EDA-cFn reactions were increased. In MGN, mesangial Ten and EDA-cFn staining was enhanced
except in segmental scars; convincing reactions were seen in cases with membranous transformation; spikes stained strongly.
In SGN, variably intense staining for Ten and all cFn isoforms was seen in glomerular necrosis, proliferation and crescents;
parietal epithelium EDA-cFn staining was noted. In DLGN, strong and extensive mesangial Ten and EDA-cFn staining was seen
as were focal EDB-and Onc-cFn reactions. Parietal cells with and without crescents stained variably with all Mabs. Obsolete
glomeruli were unreactive save for rare periglomerular Ten rims. Interstitial inflammation and fibrosis in MGN, SGN and DLGN
had moderate to strong Ten and EDA-cFn staining with rare traces of EDB-and Onc-cFn. We conclude that enhanced Ten and EDA-cFn
is a potentially reversible response to glomerular injury whereas the expression of EDB-and Onc-cFn apparently result from
necrosis and/or cellular proliferation which lead to scarring. And, while mesangial cells are the major source of these molecules,
epithelial cells might also partake in their synthesis. 相似文献
48.
M. H. N. Brumano J. L. C. Coelho E. F. Araújo D. O. Silva 《World journal of microbiology & biotechnology》1993,9(2):225-228
Optimum activity of an extracellular pectin lyase produced by Penicillium griseoroseum in submerged culture was after 120 h using 0.1% (w/v) citrus pectin as substrate. Sucrose at 0.1% (w/v) stimulated enzyme production and citrus pectin gave the highest activity of enzyme per unit growth. 相似文献
49.
P. M. Sharpe C. L. Brunet D. M. Foreman M. W. J. Ferguson 《Development genes and evolution》1993,202(3):132-143
Summary The distribution of acidic and basic fibroblast growth factors (aFGF, bFGF) was mapped during mouse embryonic palate development. Generally, they localised most intensely in the basement membrane and epithelia rather than the mesenchyme. Localisation was predominantly restricted to the palatal nasal, and medial edge epithelia. Staining was particularly intense in the medial edge epithelia at the time of mid-line epithelial seam formation. Intense staining persisted in the epithelia of the degenerating seam and later in the oral and nasal epithelial triangles. Mouse embryonic palate mesenchyme (MEPM) cells cultured in vitro on a variety of substrata (on plastic, on the surface of a collagen gel and within a collagen gel) responded to treatment with aFGF or bFGF. These responses were modulated by the culture substratum. The FGFs stimulated MEPM cell proliferation on plastic and on collagen, but inhibited cell growth in collagen. The FGFs had little effect on protein production when cells were cultured on plastic, but caused a large reduction in on-collagen and incollagen cultures. This reduction was greater in collagenous than non-collagenous proteins. Generally, treatment with FGFs stimulated the production of glycosaminoglycans (GAGs), particularly hyaluronan (HA) and dermatan sulphate (DS). In addition, the size class of HA was shifted to a higher molecular weight form. These data indicate that aFGF and bFGF may play a role in modulating mesenchymal cell matrix biosynthesis, so facilitating palatal epithelial seam degeneration.
Correspondence to: M.W.J. Ferguson 相似文献
50.
Bruno Moises de Matos Marco Augusto Stimamiglio Alejandro Correa Anny Waloski Robert 《World journal of stem cells》2023,15(5):453-465
Extracellular vesicles (EVs) are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools. Accordingly, in various biological contexts, EVs are reported to engage in immune modulation, senescence, and cell proliferation and differentiation. Therefore, EVs could be key elements for potential off-the-shelf cell-free therapy. Little has been studied regarding EVs derived from human pluripotent stem cells (hPSC-EVs), even though hPSCs offer good opportunities for induction of tissue regeneration and unlimited proliferative ability. In this review article, we provide an overview of studies using hPSC-EVs, focusing on identifying the conditions in which the cells are cultivated for the isolation of EVs, how they are characterized, and applications already demonstrated. The topics reported in this article highlight the incipient status of the studies in the field and the significance of hPSC-EVs’ prospective applications as PSC-derived cell-free therapy products. 相似文献