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1

Digitale Medien

Ultrastructural immunolocalization of actin in a fungus (1991)

Bourett, T. M. ; Howard, R. J.

Springer

Protoplasma 163 (1991), S. 199-202

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ISSN: 1615-6102

Schlagwort(e): Actin ; Freeze substitution ; Fungi ; Hyphal tip ; Immunocytochemistry ; Magnaporthe grisea

Quelle: Springer Online Journal Archives 1860-2000

Thema: Biologie

Notizen: Summary We have successfully localized fungal actin for the first time using immuno-electron microscopy and hyphal tips of the rice blast pathogenMagnaporthe grisea. Following ultrarapid freezing, samples were processed in a novel substitution fluid of 10% acrolein in anhydrous ethanol and embedded in LR White resin. A monoclonal anti-actin antibody, previously shown to recognizeM. grisea actin, bound specifically to filasomes concentrated in the peripheral cytoplasm of subapical regions, and to the core-region of the Spitzenkörper.

Materialart: Digitale Medien

URL: http://dx.doi.org/10.1007/BF01323344

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2

Digitale Medien

Brefeldin A-induced structural changes in the endomembrane system of a filamentous fungus,Magnaporthe grisea (1996)

Bourett, T. M. ; Howard, R. J.

Springer

Protoplasma 190 (1996), S. 151-163

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ISSN: 1615-6102

Schlagwort(e): Brefeldin A ; Freeze substitution ; Filamentous fungus ; Hypha ; Lectin cytochemistry ; Tip growth

Quelle: Springer Online Journal Archives 1860-2000

Thema: Biologie

Notizen: Summary Subcellular compartments comprising the endomembrane system in filamentous fungi are poorly characterized with most showing significant morphological differences from eukaryotic cells. For example, many filamentous fungi lack stacked Golgi-body cisternae, but contain “Golgi equivalents” — single cisternae or tubules which appear to serve the same functions. To help identify fungal endomembrane compartments and interrelationships between them we used a pharmacological agent, brefeldin A, known to affect specific endomembrane organelles in other organisms, most prominently the Golgi apparatus. At 10 μg/ml brefeldin A, radial hyphal growth of the rice blast pathogenMagnaporthe grisea on solid agar medium was reduced by 96% over an initial 48 h, but recovered and was reduced by only 20% over a subsequent 72 h exposure. Light microscopic examination of individual living hyphae showed that apical elongation generally halted within 1 min after exposure to brefeldin A. Acute effects of 14 μg/ml brefeldin A were characterized ultrasiructurally in cells prepared by freeze substitution. These included the appearance of two types of cisternae with unusual morphology, associated with ca. 45 nm diameter vesicles, as well as the unexpected persistence and increase in complexity of the Golgi equivalents. Also observed were (1) reduced numbers of apicale vesicles and disruption of Spitzenkörper organization, (2) apical clusters of 30–35 nm diameter microvesicles and associated tubular arrays, (3) dilation of rough endoplasmic reticulum, (4) packets of membrane-bounded electron-opaque cell wall inclusions, and (5) altered morphology of some vacuolar compartments. The distribution of concanavalin A binding sites, previously mapped to particular endomembrane compartments, was documented to aid the interpretation of these results. We conclude that brefeldin A effects on cells ofM. grisea differ from those reported with plant and animal cells, perhaps reflecting underlying differences in the endomembrane systems among these eukaryotes.

Materialart: Digitale Medien

URL: http://dx.doi.org/10.1007/BF01281314

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3

Digitale Medien

Actin in penetration pegs of the fungal rice blast pathogen,Magnaporthe grisea (1992)

Bourett, T. M. ; Howard, R. J.

Springer

Protoplasma 168 (1992), S. 20-26

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ISSN: 1615-6102

Schlagwort(e): Appressoria ; Freeze substitution ; Immunofluorescence ; Plant disease ; Phalloidin ; Pyricularia

Quelle: Springer Online Journal Archives 1860-2000

Thema: Biologie

Notizen: Summary The penetration peg is the structure used byMagnaporthe grisea to pierce the surface of rice leaves or very hard nonbiodegradable substrates. Penetration pegs produced by appressoria in vitro were examined by electron microscopy and immunofluorescence microscopy using various fluorophore labeled anti-actins. Freeze-substitution preparation of appressoria at early stages of substrate penetration showed that peg cytoplasm consisted primarily of a zone of exclusion, excluding even ribosomes, and was continuous with a similar region in the appressorium. Apical vesicles were, however, observed in short, presumably elongating pegs. Immunofluorescence microscopy was used to demonstrate binding of a monoclonal anti-actin to penetration peg cytoplasm, following “permeabilization” of appressoria by means of a brief sonication. Occasional filaments and ca. 300 nm diameter plaques were labeled in appressorial cytoplasm. Western blot analysis of germ tube extracts showed that the monoclonal probe bound predominantly to a single band with a molecular weight similar to that of rabbit muscle actin. Preincubation of the antibody with actin virtually eliminated peg labeling. We conclude that the penetration peg contains actin which may play a role in the formation of the zone of exclusion.

Materialart: Digitale Medien

URL: http://dx.doi.org/10.1007/BF01332647

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4

Digitale Medien

Fluorescence microscopy study of polymorphonuclear leukocyte substrate attached materials (1988)

Francis, Joseph W. ; Fabi, Alain Y. ; Petty, Howard R.

New York, NY : Wiley-Blackwell

Cell Motility and the Cytoskeleton 9 (1988), S. 1-8

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ISSN: 0886-1544

Schlagwort(e): substrate attached materials (SAM) ; chemotaxis ; leukocytes ; adherence ; Life and Medical Sciences ; Cell & Developmental Biology

Quelle: Wiley InterScience Backfile Collection 1832-2000

Thema: Biologie , Medizin

Notizen: We describe a technique to visualize substrate-attached materials (SAM) of polymorphonuclear leukocytes (PMN) using the fluorescent lipid analog 1, 1′-dioctadecyl-3,3,3′,3′,-tetramethylindocarbocyanine-perchlorate (DiC18Icc). DiC18Icc was incorporated into the membranes of living cells or SAMs. Since cell preparation does not require fixation, SAMs can be rapidly visualized by fluorescence microscopy. SAMs are generated by subjecting attached cells to a shearing force by rinsing with phosphate-buffered saline (PBS). The SAM-labeling protocol identified a membrane compartment as shown by detergent extraction. The SAMs of PMN leukocytes observed with this technique display complex patterns of interconnecting filaments, foci with radiating filaments, and smooth membranous areas with interconnecting filaments. The sensitivity and nondestructive nature of the DiC18Icc-labeling procedure have allowed us to observe filopodia of motile cells. The results are consistent with the hypothesis that locomotion involves a series of attachment and detachment steps. After 60 minutes of locomotion, these trailing filopodia have been measured at lengths up to 100 μm. The amount of membrane associated with these filopodia accounts for roughly 10% of the total membrane are of resting cells. These data set limits for models of membrane flow during chemotaxis.

Zusätzliches Material: 6 Ill.

Materialart: Digitale Medien

URL: http://dx.doi.org/10.1002/cm.970090102

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5

Digitale Medien

Cell surface distribution of Fc receptors II and III on living human neutrophils before and during antibody dependent cellular cytotoxicity (1989)

Petty, Howard R. ; Francis, Joseph W. ; Anderson, Clark L.

New York, NY [u.a.] : Wiley-Blackwell

Journal of Cellular Physiology 141 (1989), S. 598-605

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ISSN: 0021-9541

Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology

Quelle: Wiley InterScience Backfile Collection 1832-2000

Thema: Biologie , Medizin

Notizen: Microscopic techniques have been employed to study the cell surface distributions of the immunoglobulin Fc receptors (FcR) II and III on living human neutrophils. Fluorescein- or rhodamine-conjugated monoclonal IgG or Fab fragments directed against FcRII (CDw32) and FcRIII (CD16) were employed to label receptors. FcRII and III were found to be uniformly distributed at neutrophil surfaces during resting conditions. During neutrophil polarization and migration FcRII but not FcRIII preferentially accumulated at the uropod. Sheep erythrocytes (SRBCs) were opsonized with IgG and then incubated with neutrophils. When neutrophils were labeled prior to target addition, FcRII but not FcRIII were found to cluster at the target-effector interface. Little or no clustering of FcRs was observed if labeling was performed after target binding. SRBC oxidation was observed using Soret band illumination during transmitted light microscopy. Time-lapse studies of FcRII distribution and target oxidation were performed. FcRII formed clusters at target-effector interfaces prior to target oxidation. Three lines of evidence suggest that clustering is not a general plasma membrane response. Firstly, FcRIII do not cluster. Tannic acid-modified erythrocytes avidly bound to neutrophils but did not trigger clustering of FcRII. Furthermore, irrelevant neutrophil membrane labels were unaffected by the presence of IgG-opsonized erythrocytes. We suggest that FcRII clustering is one important component leading to the oxidative destruction of target cells.

Zusätzliches Material: 5 Ill.

Materialart: Digitale Medien

URL: http://dx.doi.org/10.1002/jcp.1041410319

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6

Digitale Medien

Influence of polysaccharides on neutrophil function: Specific antagonists suggest a model for cooperative saccharide-associated inhibition of immune complex-triggered superoxide production (1994)

Zhang, Kang ; Petty, Howard R.

New York, N.Y. : Wiley-Blackwell

Journal of Cellular Biochemistry 56 (1994), S. 225-235

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ISSN: 0730-2312

Schlagwort(e): monosaccharides ; superoxide anions ; polysaccharides ; immune complex ; β-glucan ; Life and Medical Sciences ; Cell & Developmental Biology

Quelle: Wiley InterScience Backfile Collection 1832-2000

Thema: Biologie , Chemie und Pharmazie , Medizin

Notizen: We have previously shown that certain monoisaccharides (N-acfetyl-D-glucosamine and mannose) could cooperativly inhabit the ability of neutrophils to release superoxide anions in response to immuule complexes. To test the possible orgins of the cooperative inhibition of superoxide releaqe,m we have examined the effect of a panel of particular β-glucan and hyaluronan triggered superoxide pelease from neutrophils, other polysaccharides including chitin and mannan were without effeat. Both chitin and mannan, but not other polysaccharides, inhibited the immune complex-mediated qtimulation of superoxide peleaqe in a dose-dependent fashion, In sharp contrast to the coopepative inhibition mediated by monosaacharides, chiting and mannan exhitbted Hill coefficients of 1. This inhibition of superoxide production was not due to simple blockage of Fc receotirs since fluorescent immune complexes bound equlally well to neutrophils in the presence of mannan of chitin as shown by equfluorescence microscopy and quantitative fluorometpy. Furthermore, this inhibition of superoxide release was lot observed when neutrophils were qtimulated with phorbol myristate aaetate and ionophore A23187 or Hyaluronan. Therefope, the secific inhibition of superoxide production by mannan and hitin aould lot be explailed bu eithep peceptor blockage or by some nolspecific effects on cells. We suggest that there molicules interdere with a step in transmembrace qignalling, presumably involving the intergrin CR3. The obserted Hill Cofficients suggest the possibility that one polysaccharide may simultaniouslybind to two monosaccharide bindine sites yielding a Hill coefficient of 1, wheras individual monosacaharides seperately bind vielding a Hill coefficient of 2.

Zusätzliches Material: 3 Ill.

Materialart: Digitale Medien

URL: http://dx.doi.org/10.1002/jcb.240560217

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7

Digitale Medien

Combinative ligand-receptor interactions: Epinephrine depresses RAW264 macrophage antibody-dependent phagocytosis in the absence and presence of met-enkephalin (1988)

Petty, Howard R. ; Berg, Kelly A.

New York, NY [u.a.] : Wiley-Blackwell

Journal of Cellular Physiology 134 (1988), S. 281-286

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ISSN: 0021-9541

Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology

Quelle: Wiley InterScience Backfile Collection 1832-2000

Thema: Biologie , Medizin

Notizen: Macrophages have been shown to possess cell surface receptors for opiates and catecholamines. The abilities of these ligands to affect RAW264 macrophage antibody-dependent effector activity directed against sheep red blood cells were tested. Phagocytosis was measured by the uptake of 51Cr labeled erythrocytes and optical microscopy. Cytolysis was measured by 51Cr-release assays. Met-enkephalin increased specific antibody-dependent phagocytosis in a dose-dependent fashion; the optimal dose was found to be 10-8 M. Epinephrine diminished phagocytosis in a dose-dependent manner exhibiting a maximal inhibition at 10-4-10-5 M. This inhibition can be blocked by propranolol. The combined effects of simultaneous treatment with met-enkephalin and epinephrine were measured. At the several doses tested, the combined effects of these two ligands on the amount of phagocytosis were equivalent to or more inhibitory than epinephrine alone. Thioglycolate-elicited murine peritoneal macrophages demonstrated similar responses to epinephrine, met-enkephalin, and their combination. Therefore, in vitro models more closely approximating in vivo neuroregulation of macrophage function demonstrate phagocytic inhibition.

Zusätzliches Material: 4 Ill.

Materialart: Digitale Medien

URL: http://dx.doi.org/10.1002/jcp.1041340215

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8

Digitale Medien

Neutrophil C3bi receptors: Formation of membrane clusters during cell triggering requires intracellular granules (1987)

Petty, Howard R. ; Francis, Joseph W. ; Todd, Robert F. ; [weitere]

New York, NY [u.a.] : Wiley-Blackwell

Journal of Cellular Physiology 133 (1987), S. 235-242

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ISSN: 0021-9541

Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology

Quelle: Wiley InterScience Backfile Collection 1832-2000

Thema: Biologie , Medizin

Notizen: Video-intensification fluorescence microscopy has been used to study the cell surface distribution of the complement receptor (CR) for C3bi (CR3) on human neutrophils. Fluorescin- or rhodamine-labeled monoclonal IgG or Fab fragments of antireceptor antibody were used as probes of receptor localization. C3bi receptors are uniformly distributed on untreated cells. Glass coverslips were coated with lipopolysaccharide (LPS) and serum was added; the serum deposits complement components, including C3bi, on the surface. When neutrophils were adherent to these coverslips, receptors were found in large clusters, and a fraction of the fluorescence remained uniform. Double-labeling studies were conducted by first labeling with anti-CR3 followed by attachment to LPS/serum-treated slides. This, in turn, was followed by labeling with the antibody conjugated to a second fluorophore. These studies revealed that the CR3 clusters were predominantly new antigenic sites exposed after attachment to the LPS/serum-treated slides. To determine the contribution of granule-associated CR3, we have studied neutrophils defective in receptor up-regulation, neutrophil cytoplasts, and a stimulator of granule release, A23187. Neutrophils from a patient with specific granule deficiency were found to be defective in granular CR3 and did not form clusters on C3-modified surfaces. The patient's neutrophils were defective in CR3 up-regulation and enzyme release as shown by fluorescence flow cytometry and gelatinase release, respectively. Cytoplasts also failed to show CR3 clusters on LPS/serum-treated coverslips. Furthermore, neutrophils treated with A23187 demonstrated numerous CR3 clusters. We suggest that formation of CR3 membrane domains during immune recognition requires the participation of intracellular granules. We speculate that these domains are formed by fusion of CR3-bearing granules at local sites of adhesion.

Zusätzliches Material: 5 Ill.

Materialart: Digitale Medien

URL: http://dx.doi.org/10.1002/jcp.1041330206

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9

Digitale Medien

Combinative ligand-receptor interactions: Effects of CAMP, epinephrine, and met-enkephalin on RAW264 macrophage morphology, spreading, adherence, and microfilaments (1989)

Petty, Howard R. ; Martin, Susan M.

New York, NY [u.a.] : Wiley-Blackwell

Journal of Cellular Physiology 138 (1989), S. 247-256

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ISSN: 0021-9541

Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology

Quelle: Wiley InterScience Backfile Collection 1832-2000

Thema: Biologie , Medizin

Notizen: Cell surface ligand-receptor interactions play a central role in the regulation and expression of macrophage function. Included among these macrophage membrane receptors are the β-adrenergic and opioid receptors. We studied the abilities of epinephrine, met-enkephalin, forskolin, and adenosine 3′:5′ cyclic monophosphate (cAMP) analogues to affect macrophage morphology, spreading, and adherence. Cell spreading was quantitated by measuring the perimeters of adherent cell images recorded by videomicroscopy. Epinephrine induced a dose-dependent decrease in macrophage spreading; at 10-5 M epinephrine the mean perimeter was 10.4 ± 0.3μm in comparison to 15.0 ± 1.0 μm for controls. The inhibition of spreading can be blocked by the antagonist propranolol. On the other hand, met-enkephalin induced a dose-dependent increase in macrophage spreading, with a perimeter of 18.5 ± 1.0 μm at 10-8 M. Since catecholamines and opioids are simultaneously released from chromaffin cells of the adrenal, we examined the combinative effects due to treatment with both ligands. When macrophages were exposed to 10-5 M epinephrine and 10-8 M met-enkephalin, cell morphology and spreading were indistinguishable from that due to 10-5 M epinephrine alone. The epinephrine dose-response curve in the presence of 10-8 M met-enkephalin, was similar to that of epinephrine alone. The β-adrenergic receptor is apparently capable of diminishing or abrogating the opioid receptor signal(s). These combinative and epinephrine-mediated effects may be at least partially accounted for by the action of cAMP. Forskolin and the cAMP analogues N6 -2′-O-dibutyryladenosine 3′:5′ cyclic monophosphate (dbcAMP) and 8-bromoadenosine 3′:5′ cyclic monophosphate (Br-cAMP) affected cell morphology and spreading in the same fashion as epinephrine. These differences in morphology and spreading behavior were accompanied by changes in the distribution of F-actin, as judged by phalladicin staining and fluorescence microscopy. We suggest that cAMP and microfilaments play important roles in receptor-mediated neuroregulation of macrophage function.

Zusätzliches Material: 10 Ill.

Materialart: Digitale Medien

URL: http://dx.doi.org/10.1002/jcp.1041380205

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10

Digitale Medien

G2 + M arrest of cultured mammalian cells after incorporation of tritium-labeled nucleosides (1977)

Marz, R. ; Zylka, J. M. ; Plagemann, P. G. W. ; [weitere]

New York, NY [u.a.] : Wiley-Blackwell

Journal of Cellular Physiology 90 (1977), S. 1-8

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ISSN: 0021-9541

Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology

Quelle: Wiley InterScience Backfile Collection 1832-2000

Thema: Biologie , Medizin

Notizen: Novikoff rat hepatoma cells were propagated in suspension cultures containing 0.5 to 10 μC of 3H-methyl-thymidine, 3H-5-uridine, 3H-G-adenosine or 3H-8-adenine. The presence of the 3H-labeled precursors caused an inhibition of cell replication which was due to a delay or arrest of the cells in G2 and M. The degree of inhibition was proportional to the amount of radioactivity incorporated into nucleic acids. Almost immediate and complete inhibition resulted from incubation with 10 μC 3H-thymidine/ml. The presence of 0.5 μC 3H-thymidine/ml caused a significant increase in the relative proportion of cells in G2 + M, even though the population doubling time of the culture appeared to be unaltered.

Zusätzliches Material: 5 Ill.

Materialart: Digitale Medien

URL: http://dx.doi.org/10.1002/jcp.1040900102

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