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  • 1
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    Stuttgart : Thieme
    Type of Medium: Unknown
    Pages: 528 S.
    Edition: 3., neu bearb. Auflage
    ISBN: 3131065133
    Language: German
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  • 2
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Annexins were isolated fromParamecium cell homogenates by standard ethylene glycol tetraacetic acid (EGTA) extraction and 100 000-g centrifugation. Two different antibodies (Abs) against synthetic peptides were used, Call-15 and B15, which in mammalian cells recognize a sequence of annexin II or a common sequence occurring in several annexins (except for annexin II), respectively. With anti-Call-15 Abs, western blots from EGTA extracts showed strongly reactive bands of 44.5 and 46 kDa and of higher values. Some of these bands bound to the 100 000-g pellet fraction when Ca2+ was added. Immuno- and affinity labelling revealed selective. Ca2+-dependent labelling of the cell cortex, with enrichent around trichocyst docking sites (facing subplasmalemmal Ca2+ stores). Cortical fluorescence labelling decreased in wild-type (7S) cells when trichocyst ghosts were detached after synchronous exocytosis. Similarly, cortical labelling was reduced when intact trichocysts were detached from the cell surface of non-discharge mutant cells (nd9–28°C, showing identical bands on blots), which then contained numerous heavily labelled phagolysosomes. This strongly suggests annexin downregulation. All together, the dynamic labelling of cortical structures we observed strongly supports involvement of calpactin-like annexins in trichocyst docking. Anti-B15 Abs recognized a band of 51 kDa and some of higher values. These Abs selectively labelled the outlines of the cytoproct, the site of spent phagolysosome exocytosis. In conclusion, our data indicate involvement of specific sets of annexins in site-specific positioning and attachment of widely different secretory organelles at the cell surface inParamecium cells.
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  • 3
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In Paramecium cells Ca++-stimulated triggering of the exocytosis of secretory vesicles (“trichocysts”) was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some “resting” trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the “inner lamellar sheath” from where deposits seemed to “radiate” into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those “resting” trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca++-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possibly in combination with the sudden activation of an ATPase systemlocalized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca++ and then fixed with OsO4 plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including “ciliary granule plaques”) also contained Ca, P and S. Cells contain osmiophilic “calcium-storing vacuoles” which were selectively rich in Ca and S but devoid of P.
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  • 4
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In paramecia the membranes of alveoli and trichocysts are permanently connected to the cell membrane by membrane-junctions, which consist of membrane-intercalated particles in a regular geometrical arrangement. Trichocysts contain secretory material discharged by exocytosis. In unfixed or fixed cells these two compartments were impermeable to the following tracers: To “microperoxidases”, i.e. a cytochrome c-derived heme-nonapeptide and a heme-undecapeptide (WM∼1650, 1900) applied in vivo, as well as to lanthanum and cytochrome c used during (La) or after (cytochrome c) fixation. The heme-nonapeptide was prepared by TPCK-trypsin digestion of cytochrome c and subsequent purification by Sephadex gel chromatography—a simple and inexpensive new procedure resulting in preparations of high yield and purity. Tracers entered alveoli only when the plasmalemma and the alveolar membranes ruptured upon glutardialdehyde fixation. In no case were transmembraneous channels detectable in regions containing membrane-intercalated particles; this holds true for all tracers used and for freeze-fracture replicas obtained by tantalum-tungsten evaporation. With regard to attachment sites over trichocysts our results do not support the assumptions by others according to which exocytosis would be driven by an osmotic shift via transmembraneous channels (which would be analogous to inter-cellular coupling phenomena mediated by gap-junctions), unless such channels would be assumed to operate as carriers rather than via diffusion. Tracers did not penetrate trichocysts before exocytosis occurred. The functional role of membrane-intercalated particles on trichocyst attachments remains nuclear. Despite some resemblance with gap-junctions all types of intra-cellular membrane-junctions investigated are functionally “tight” at the level of “resolution” obtained with tantalum-tungsten-shadowing and with the tracers used.
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  • 5
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary A heme-nonapeptide (H-9-P)1, applicable to electron microscopic cytochemistry via peroxidase-like activity, was prepared by passing horse heart cytochrome c through a column with Sepharose and covalently attached trypsin. After purification by column chromatography (Sephadex G50 Superfine, Biogel P-2) a maximal yield of ∼50% and purity of 〉99% was achieved. A concise schedule allows for inexpensive preparation of H-9-P with standard laboratory equipment. H-9-P has the following properties: Its structure is (14) Cys-Ala-Gln-Cys-His-Thr-Val-Glu-Lys (22) with heme attached to Cys (14) and (17). MW=1630, pI=4.95, λ E(max) pH 7 = 397.5 nm, ε 22 °C, pH 7 397.5 nm = 1.11 × 105 [Liter/Mole x cm]. With the use of a diaminobenzidine-H2O2-medium — as applied for cytochemistry — we determined spectrophotometrically a pHopt=12.5 and an apparent K5 = 3.14 × 10− 3 [M]. Glutardialdehyde leads to considerable de-activation and, according to SDS-polyacrylamide-gel-electrophoresis, to diffuse crosslinking accompanied by a shift of the active pH-region towards neutral pH values. An attempt was made to optimize the cytochemical assay. The peroxidase-like activity of H-9-P is well comparable to that of other heme-tracers; only horseradish peroxidase has a higher turnover number. When injected to mice or added to cell suspensions, even high concentrations of H-9-P did not entail any signs of toxicity.
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  • 6
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Group
    Nature 252 (1974), S. 722-724 
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Exocytosis, monitored using the phase microscope, was triggered by enhancing transmembraneous ion flux via cationophores, that is, small molecules incorporated spontaneously into membranes. Intramembraneous morphological changes were analysed by freeze-fracture. lono-phores used were X-537 A ...
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  • 7
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have followed the time-dependent transfection of Paramecium cells with a vector containing the gene of green fluorescent protein (GFP) attached to the C-terminus of the PtSERCA1 gene. The outlines of alveolar sacs (ASs) are labelled, as is the endoplasmic reticulum (ER) throughout the cell. When GFP fluorescence is compared with previous anti-PtSERCA1 antibody labelling, the much wider distribution of GFP (ER+ASs) indicates that only a small amount of SERCA molecules is normally retained in the ER. A second isoform, PtSERCA2, also occurs and its C-terminal GFP-tagging results in the same distribution pattern. However, when GFP is inserted in the major cytoplasmic loop, PtSERCA1 and two fusion proteins are mostly retained in the ER, probably because of the presence of the overt C-terminal KKXX ER-retention signal and/or masking of a signal for transfer into ASs. On the overall cell surface, new SERCA molecules seem to be permanently delivered from the ER to ASs by vesicle transport, whereas in the fission zone of dividing cells ASs may form anew. In cells overexpressing PtSERCA1 (with C-terminal GFP) in ASs, [Ca2+]i regulation during exocytosis is not significantly different from controls, probably because their Ca2+ pump has to mediate only slow reuptake.
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  • 8
    ISSN: 1573-4935
    Keywords: Electron microscopy ; secretion ; neuropeptides ; exocytosis ; endocytosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Exo- and endocytotic processes induced by depolarization of isolated neurosecretory nerve terminals show a close temporal correlation, which suggests a short time of integration of the neurosecretory granule membrane with the plasma membrane. In order to determine minimal time requirements for exocytosis-coupled endocytosis to occur, we have analyzed by electron microscopy uptake of horserdish peroxidase (HRP) as a fluid phase marker at the onset of depolarization. We have applied rapid mixing and sampling (quenched flow) to assess events in subsecond time peroids after stimulation. A significant number of labelled endocytotic vacuoles was observed during the first second of depolarization. This number then further increased by a factor of about 2 (within 5 s) and 4 (within 50s). Thus, as for exocytosis, the rate of endocytosis decreased considerably during prolonged stimulation. These data indicate i) that a substantial proportion of secretory granules undergoes exocytosis very shortly after stimulation, and ii) that, following exocytosis, the minimal time required for consecutive membrane retrieval is in the sub-second range.
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  • 9
    ISSN: 1432-0878
    Keywords: Protein secretion ; Defensive glands ; Blatta orientalis ; Cytology ; Autoradiography ; Insekts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Description / Table of Contents: Zusammenfassung Im Bereich der abdominalen Tergite V-X und oberseits and den Zerzi liegt bei Nymphen beider Geschlechter und bei adulten Weibchen von Blatta orientalis statt des einschichtigen Epithels ein zweischichtiges Drüsengewebe vor, welches ein visköses Sekret aus Wasser, freien Aminosäuren (+ Glutamin), Oligo- und zahlreichen Polypeptiden auf die Tergitenoberfläche sezerniert. Die strukturelle Differenzierung des Drüsengewebes ist mit der Sekretionsaktivität korreliert, sowohl während der Ontogenese als auch im Bereich verschiedener Tergite (Maxima: weibliche Subimagines, Tergite VI und VII). Untersuchungen mittels hochauflösender quantitativer Autoradiographie ergaben, daß injizierte Aminosäuren im größten Teil der Drüsenzellen angereichert werden: Markierte Zellen zeigen Radioaktivität im reichlich ausgebildeten rauhen endoplasmatischen Retikulum, Golgi-Apparat, in Sekretgranula und in ihrem Endapparat. Dieser durchsetzt die Drüsenzelle als langer gewundener Kanal mit Bürstensaum, in welchen je eine darüberliegende Gangzelle einen chitinösen Ausführgang inseriert. Die gleichzeitige Anwesenheit von markierten und nicht markierten Zellen mit praktisch gleich stark entwickeltem endoplasmatischem Retikulum und Golgi-Apparat zeigt, daß die morphologische Ausbildung dieser mit der Proteinsekretion befaßten Organellen nicht unbedingt deren Aktivität reflektiert. Vereinzelt stehen Zellfortsätze mit den morphologischen Charakteristika neurosekretorischer Tätigkeit in direktem Kontakt mit Drüsenzellen. Eine Abwehrfunktion des viskösen Sekrets durch bloße mechanische Behinderung kleiner räuberischer Arthropoden wurde sichergestellt, wobei es dem Beutetier gelingt, zu flüchten. Weiters wurde ein zweiter Drüsenzelltyp beobachtet, der mit injizierten Aminosäuren nur schwach markierbar ist, ebenfalls einen Endapparat besitzt, jedoch arm an rauhem endoplasmatischem Retikulum und gleichzeitig reich an Mitochondrien, Golgi-Apparaten und kleinen Vesikeln ist. Die Funktion dieses zweiten Zelltyps ist zwar nicht sichergestellt, möglicherweise reguliert er jedoch die funktionell wichtige Viskosität des Sekretes.
    Notes: Summary A two-layered glandular tissue occurs on tergites V to X and on the cerci of juvenile specimens of both sexes and of adult females of Blatta orientalis, in place of the usual monolayer of epidermal cells. This gland tissue contains two cell types and secretes a viscous product of water, free amino acids (+ glutamine), oligo- and several polypeptides onto the tergal surface. The structural differentiation of the gland is correlated with secretory activity, both in different molting stages and in different tergites of an individual; maximal values are found in tergites VI und VII on last instar females. Applying quantitative radioautography on the electron microscope level, we found, that although the most common gland cell type contained an abundantly developed rough endoplasmic reticulum and Golgi-apparatus, characteristic of protein secreting cells, not all of them incorporated equally the injected amino acids. This is consistent with an asynchronous secretory cycle, also suggested by biochemical studies. Of great significance is the demonstration that the fine structural elaboration of the cellular organelles involved in protein synthesis cannot be used as a criterion for their ongoing activity. The secretion is discharged into an end-apparatus consisting of a tortuous canal with a brushborder that penetrates the whole gland cell. One unbranched chitinous duct, formed by a “duct carrying cell”, is inserted into the end-apparatus of each gland cell. Occasionally, cell processes exhibiting the typical morphological characteristics of neurosecretory cells are seen in direct contact with gland cells. A defensive function of the secretion which acts by mechanically impairing smaller predatory arthropods was ascertained. To achieve this effect and to allow the preyanimal to escape, the secretion has to be adjusted to a proper viscosity by an adequate dilution. This might be achieved by the second gland cell type, which was not selectively labelled by injected amino acids; this cell type contains an endapparatus, abundant mitochondria, Golgi-apparatuses and small vesicles, but only few profiles of rough endoplasmic reticulum.
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  • 10
    ISSN: 1059-910X
    Keywords: C1q ; Calcium ; Specific binding ; SP-A ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: We analyzed the binding mechanism of human recombinant lung surfactant protein A (SP-A) to rat alveolar macrophages using anti-SP-A antiserum and protein A coated onto gold particles. Results were compared with our recent data on binding and uptake of SP-A-coated colloidal gold particles. The rationale for the current approach was to avoid any possible steric effects on SP-A binding to the cell surface. Binding of unlabeled SP-A depends on the presence of calcium ions in the medium and involves a mannose-specific mechanism. Binding is partly inhibited by the collagenase-resistent fragment of SP-A, representing mainly the globular part of SP-A. Taken together, these facts indicate binding of SP-A via the carbohydrate binding site on the globular region of SP-A. On the other hand, a partial inhibition of SP-A binding by fragments of C1q (representing the collagenous region of C1q) indicates a second binding site for SP-A by the collagen-like portion to the C1q receptor of macrophages. We conclude that two different mechanisms are probably involved in SP-A binding to alveolar macrophages. Specificity of the binding was shown with fluorescein-labeled SP-A. Binding was inhibited by an excess of unlabeled SP-A. Binding and uptake of SP-A are seen only with alveolar macrophages and not with other macrophage populations isolated from rat, such as liver macrophages (Kupffer cells), resident peritoneal macrophages, and peritoneal macrophages activated by Corynebacterium parvum. Therefore, binding sites for SP-A occur exclusively on alveolar macrophages. In addition, the intracellular Ca2+ concentration of the lung macrophages was determined by using the fluorescent dye fura-2/AM. Intracellular [Ca2+] increased immediately after addition of SP-A. This indicates immediate activation of macrophages by SP-A. © 1993 Wiley-Liss, Inc.
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