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G Knoll

Publications and source records attributed to G Knoll.

36 records · Page 2Linked to original sources
PubMed1992

A rapid calcium influx during exocytosis in Paramecium cells is followed by a rise in cyclic GMP within 1 s.

The synchrony of trichocyst exocytosis in Paramecium allows temporal correlation of associated events. Using quenched flow we observed a Ca2+ influx concurrent with exocytosis within 80 ms after stimulation with the secretagogue aminoethyldextran. Cyclic AMP did not change in depency of stimulation. Cyclic GMP transiently increased after 500 ms, culminating at 2 s, and thus considerably lags behind exocytosis induction and influx of Ca2+. Both Ca2+ influx and rise in cGMP are known to be induceable also by Ba2+ or veratridine, allegedly via the opening of ciliary Ca2+ channels. However, only veratridine stimulated exocytosis. We conclude that both aminoethyldextran and veratridine induce an exocytosis-associated Ca2+ influx, which is responsible for the rise in cGMP, through an as yet unknown pathway.

Animals↗
PubMed1992

Quantitative immuno-gold labelling and ultrastructural preservation after cryofixation (combined with different freeze-substitution and embedding protocols) and after chemical fixation and cryosectioning. Analysis of the secretory organelle matrix of Paramecium trichocysts.

Among the variety of parameters affecting immuno-gold labelling efficiency, mainly the effects of different preparative protocols were tested. Preservation of ultrastructure and of antigenicity are the salient features of this study. We have labelled insoluble components of the secretory matrix of Paramecium trichocysts with specific antisera, using 10 nm colloidal gold particles. The highest labelling efficiency was obtained with fast freezing (cryofixation, either by sandwich or spray-freezing), freeze-substitution in methanol (without added fixatives) and hydrophilic Lowicryls, particularly when applied at low temperatures (K11M at 193 K). The presence of different chemical fixatives always reduced the labelling density and some recommendations from the literature do not appear advisable. Methods commencing with fixation at greater than or equal to 0 degree C, such as "progressive lowering of temperature" (PLT) or preparation of cryostat sections, i.e. with chemical pretreatments, always resulted in lower labelling density. Our data appear, therefore, relevant for optimal immuno-gold labelling of insoluble antigens and emphasize the potential of cryofixation as a primary preparation step. In addition, ultrastructural preservation was also superior after cryofixation.

Acrylic Resins↗
PubMed1992

Exo-endocytosis in isolated peptidergic nerve terminals occurs in the sub-second range.

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 horseradish 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 sub-second time periods 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 50 s). 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.

Animals↗
PubMed1992

Intracellular calcium during chemotaxis of Dictyostelium discoideum: a new fura-2 derivative avoids sequestration of the indicator and allows long-term calcium measurements.

During stimulation of Dictyostelium discoideum amoebae with the chemoattractant cAMP, extracellular calcium is taken up by the cells. The aim of this study was to determine the cytosolic free calcium concentration ([Ca++]i) during chemotaxis of Dictyostelium cells. In contrast to most vertebrate cells, three major drawbacks were encountered: 1) the indicator fura-2 could not be introduced into the cells by incubation with the ester form, 2) once loaded, the dye was rapidly sequestered into vesicles, 3) the organic anion transport blocker probenecid was not suitable to block sequestration. These problems were met by introducing the indicator into the cells with the scrape-loading technique adapted for use with Dictyostelium and the construction of a new fura-2 derivative, fura-2-dextran. Scrape-loading of Dictyostelium yielded up to 40% of labeled, vital cells. Fura-2-dextran fulfilled the following criteria: 1) it remained homogeneously distributed in the cytoplasm of motile Dictyostelium cells, 2) it retained the fluorescence intensity of fura-2 and the affinity for calcium binding, 3) it was very well suitable to demonstrate changes of [Ca++]i in serum-stimulated fibroblasts. [Ca++]i-measurements with fura-2-dextran in chemotactically active D. discoideum amoebae revealed that the large decrease in the extracellular calcium concentration is not accompanied by an overall change in [Ca++]i. Chemotaxis in this organism occurs in the absence of global changes in [Ca++]i. However, we cannot exclude either short-lived or local changes just beneath the plasma membrane.

Animals↗
PubMed1991

Effect of intracoronary diltiazem on infarct size and regional myocardial function in the ischemic reperfused canine heart.

This study was designed to investigate whether intracoronary diltiazem given before reperfusion could enhance myocardial salvage in the canine heart. Twenty-five dogs were subjected to 90 min of coronary occlusion followed by 4 h of reperfusion. The dogs were assigned to one of three experimental groups. The early diltiazem group received intracoronary diltiazem into the distal coronary bed at the onset of coronary occlusion and for 60 min after reperfusion. The late diltiazem group received the same amount of drug beginning 15 min before reperfusion and the control group received saline solution for 90 min of occlusion and 60 min of reperfusion. Infarct size expressed as a percent of the area at risk was significantly smaller in the early and late diltiazem groups (15.6 +/- 3.6% and 21.2 +/- 5.1%, respectively) than in the control group (49 +/- 4.6%) (p less than 0.05). Intracoronary diltiazem restored systolic function of the stunned, previously ischemic tissue to essentially normal preocclusion values. Segmental shortening after reperfusion averaged 21.6% in the early diltiazem group versus 0 +/- 1.7% and 7.3 +/- 4% for the control and late diltiazem groups, respectively (p less than 0.05). Low dose intracoronary diltiazem did not alter hemodynamic variables or myocardial blood flow but did improve segmental shortening 2 and 6 h after reperfusion. These data indicate that intracoronary diltiazem given during occlusion or just before reperfusion increases the salvage of myocardium compared with the salvage achieved by reperfusion alone. These results also suggest that intracoronary diltiazem given during the ischemic period enhances systolic contractile function of postischemic stunned myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Quenched flow analysis of exocytosis in Paramecium cells: time course, changes in membrane structure, and calcium requirements revealed after rapid mixing and rapid freezing of intact cells.

Synchronous exocytosis in Paramecium cells was analyzed on a subsecond time scale. For this purpose we developed a quenched flow device for rapid mixing and rapid freezing of cells without impairment (time resolution in the millisecond range, dead time approximately 30 ms). Cells frozen at defined times after stimulation with the noncytotoxic secretagogue aminoethyldextran were processed by freeze substitution for electron microscopic analysis. With ultrathin sections the time required for complete extrusion of secretory contents was determined to be less than 80 ms. Using freeze-fracture replicas the time required for resealing of the fused membranes was found to be less than 350 ms. During membrane fusion (visible 30 ms after stimulation) specific intramembranous particles in the cell membrane at the attachment sites of secretory organelles ("fusion rosette") disappear, possibly by dissociation of formerly oligomeric proteins. This hitherto unknown type of rapid change in membrane architecture may reflect molecular changes in protein-protein or protein-lipid interactions, presumably crucial for membrane fusion. By a modification of the quenched flow procedure extracellular [Ca++] during stimulation was adjusted to less than or equal to 3 x 10(-8) M, i.e., below intracellular [Ca++]. Only extrusion of the secretory contents, but not membrane fusion, was inhibited. Thus it was possible to separate both secretory events (membrane fusion from contents extrusion) and to discriminate their Ca++ requirements. We conclude that no Ca++ influx is necessary for induction of membrane fusion.

Animals↗
PubMed1991

A preparation method of specimens of the fungus Penicillium chrysogenum for ultrastructural and immuno-electron microscopical studies.

A combination of cryofixation without pre-treatment, freeze-substitution and low-temperature embedding was used to prepare specimens of Penicillium chrysogenum for electron microscopy. To produce specimens which are thin enough for appropriate cryofixation, the P.chrysogenum colonies were grown between dissected-dialysis tubing on an agar plate, which in addition allowed longitudinal sectioning. In contrast to classical chemical fixation, this preparation procedure resulted in excellent preservation of ultrastructure. Furthermore, the penicillin biosynthetic enzyme acyltransferase could be unequivocally located by immunogold labelling, indicating a preservation of antigenic properties of the specimen. Labelling density was not conspicuously affected when using different freeze-substitution media, but it was reduced after embedding in Epon 812.

Acyltransferases↗
PubMed1988

Reconstitution of Na+/K+-ATPase into phosphatidylcholine vesicles by dialysis of nonionic alkyl maltoside detergents.

The reconstitution of Na+/K+-ATPase from outer medulla of rabbit kidney into large unilamellar liposomes was achieved through detergent removal by dialysis of mixed micellar solutions of synthetic dioleoyl phosphatidylcholine/octyl glucoside and Na+/K+-ATPase/decyl maltoside or decenyl maltoside. Tight, transport-active liposomes were formed when the lipid and the enzyme were solubilized separately in the nonionic detergents and mixed immediately before starting the dialysis. The two maltoside detergents with different structures of the hydrophobic part of the molecule proved to be well suited for the solubilization of Na+/K+-ATPase with high retention of enzyme activity; the inactivation of enzyme being evidently slower with the unsaturated decenyl maltoside. The diameters of the proteoliposomes, 110 and 170 nm, respectively, were also dependent on the structure of the maltoside detergent, the saturated decyl maltoside producing the bigger liposomes. After freeze-fracture, both preparations exhibited intramembranous particles as structural indicators of successful reconstitution. The electrogenic activity of the reconstituted enzyme was determined by fluorescence measurements with Oxonol VI and by tracer-flux measurements with 22Na+.

Animals↗
PubMed1988

Influenza virus-model membrane interaction. A morphological approach using modern cryotechniques.

The membrane fusion activity of influenza virus was characterized morphologically using a model system composed of a highly purified influenza B virus suspension and ganglioside-containing zwitterionic liposomes. Electron microscopical analysis was performed after a combination of fast-freezing with either freeze-fracture or freeze-substitution-thin sectioning, ensuring maximal time resolution and avoiding preparation artifacts. In a parallel fluorescence 'lipid mixing' fusion assay, influenza virus-membrane fusion was characterized biochemically. Biochemical and morphological data are in full agreement, indicating negligible membrane fusion activity at neutral pH and high fusion activity at low pH. The freeze-fracture morphology strongly suggests a local point contact between viral and liposomal membrane at neutral pH, and a local point fusion mechanism for influenza virus-membrane fusion upon lowering of the pH. Fusion is followed by lipid mixing, lateral diffusion of viral spike proteins and exposure of viral contents at the inner liposomal surface.

Freeze Fracturing↗
PubMed1988

Fusion of liposomes with the plasma membrane of epithelial cells: fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections.

The fusion of liposomes with the plasma membrane of influenza virus-infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry. 24:3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apical and basolateral plasma membrane domains after fusion was assessed by autoradiography of liposomal [3H]dipalmitoylphosphatidylcholine after rapid freezing or chemical fixation and further processing by freeze substitution and low temperature embedding. Before fusion, radioactivity was solely detected on the apical cell surface, indicating the absence of redistribution artifacts and demonstrating the reliability of lipid autoradiography on both a light and electron microscopical level. After induction of fusion by a low pH treatment, the basolateral plasma membrane domain became progressively labeled, indicative of rapid lateral diffusion of [3H]dipalmitoylphosphatidylcholine in the plasma membrane. Analysis of individual fusion events by freeze fracture after rapid freezing confirmed the rapid diffusion of the liposomal lipids into the plasma membrane, as intramembrane particle-free lipid patches were never observed. After the induction of liposome-cell fusion, well-defined intramembrane particles were present on the otherwise smooth liposomal fracture faces and on the fracture faces of the plasma membrane. Morphological evidence thus was obtained in favor of a local point fusion mechanism with an intramembrane particle as a specific structural fusion intermediate.

Animals↗
PubMed1987

Ultrastructural analysis of dynamic cellular processes: a survey of current problems, pitfalls and perspectives.

Dynamic phenomena in cells that can be analyzed on the ultrastructural level comprise so different aspects as ion shifts, conformational changes of macromolecules, membrane particle rearrangements, lipid phase transitions, protein-protein interactions (notably ligand-receptor interactions, including their sorting and sequestration), reversible membrane-to-membrane contacts, membrane fusions, transcellular transport phenomena, restructuring of cytoskeletal elements, ciliary and flagellar beat, cell shape changes, etc. Only some of these phenomena can be analyzed under stationary conditions, while others are unidirectional and sometimes very rapid. Therefore, the methodical approaches to be used (primary methods and follow-up procedures) might be widely different. Quite different methods are available, such as fast freezing, specific labeling, low temperature processing and/or analysis, x-ray-microanalysis, etc. Only occasionally are there alternative non-ultrastructural control methods available. This survey paper tries to analyze the degree of reliability (or uncertainty) of current methods and to pinpoint the goals and eventually also new methodical perspectives for an integrative approach to analyze dynamic cellular processes with the high temporal and spatial resolution provided by the electron microscope.

Animals↗
PubMed1985

The regulation of mitochondrial-bound hexokinases in the liver.

A functional coupling between bound hexokinase and the inner mitochondrial compartment has been shown. It is based structurally on the binding of hexokinase to a pore protein which is present in zones of contact between the two boundary membranes. The latter was observed by electron microscopic localization of antiporin and hexokinase at the mitochondrial surface. The four isoenzymes present in liver differ considerably in their activity after binding to the mitochondrial surface. This was found by binding studies using the four isoenzymes isolated from the supernatant. Isoenzyme IV did not bind at all. Isoenzymes I-III did bind and became activated: I, 5.9-fold; II, 39-fold; and III, 1.3-fold. These results suggest that the in vivo activity of hexokinase in the mitochondrial fraction is much larger than so far observed. Furthermore the binding of isoenzymes was differently affected by metabolites. Glucose-6-phosphate exclusively desorbed isoenzyme I from the mitochondrial membrane whereas free fatty acids predominantly liberated isoenzymes II and III. A reciprocal change of the levels of free fatty acids and glucose 6-phosphate in livers of starved rats therefore, can explain why exclusively mitochondrial-bound isoenzymes II and III decreased 10-fold while at the same time isoenzyme I increased.

Animals↗
PubMed1984

Alterations in liver mitochondrial function as a result of fasting and exhaustive exercise.

The effect of exercise upon liver mitochondria structure and function was examined in fasted and fed rats, following a single run to exhaustion on a motor-driven treadmill. Exercise alone and exercise coupled with fasting both produced a significant decrease in the amount of hexokinase bound to the mitochondria, as well as reduction in the ADP/O ratio and acceptor control index measured in the presence of succinate. The mitochondria of the exercised animals, when exposed to freeze-fracture analysis while in state 3, displayed fewer deflections in the fracture plane between the inner and outer membrane than those isolated from control animals. This suggests that fewer contacts existed between the two membranes. Measurements based upon the binding of 8-anilinonaphthalene 1-sulphonate indicated that there was an increase in the net negative charge on the surface of the mitochondrial membranes of the exercised animals. All of these effects could be mimicked by incubation of mitochondria from control animals with free fatty acids. This fact, coupled with the observation that washing of the mitochondria with a solution comprising 5% (w/v) albumin could reverse all of the consequences of exercise, suggests that these alterations in mitochondrial structure and function may be the result of the increase in plasma free fatty acids that accompanies long-term exercise. Furthermore, the observation that the exercise-induced changes are dynamic and readily reversible indicates that the mitochondria were not necessarily damaged, but rather that the coupling of oxidative phosphorylation may be subject to physiological regulation.

Anilino Naphthalenesulfonates↗
PubMed1983

Changes in freeze-fractured mitochondrial membranes correlated to their energetic state. Dynamic interactions of the boundary membranes.

Structural changes of mitochondria in correlation to their energetic state have been observed as matrix expansion and condensation. In this communication we describe a morphological correlation in freeze-fractured mitochondrial membranes which is also dependent on the metabolic state of the organelle: the frequency by which the fracture plane following the inner or outer boundary membrane deviates by jumping from one membrane to the other is higher in phosphorylating mitochondria when compared to freshly isolated or energized mitochondria. These deflections of the fracture plane occur mostly in minimal, short steps showing close apposition of the two boundary membranes. We therefore conclude that the observed change in morphological appearance is produced by a change in interactions between the inner and outer membranes correlated to the different functional states of the inner membrane.

Animals↗
PubMed1980

[Suppression of bone marrow in neonatal herpes simplex virus infection under virustatic therapy with adenine arabinoside-5'-monophosphate (author's transl)].

The case of a generalized neonatal herpes-simplex-virus infection with lethal outcome is presented. Therapy with adenine arabinoside-5'-monophosphate (ARA AMP) was employed with a good virustatic effect. Suppression of bone marrow and a possible immunological suppression was seen at low dose level already. Therefore in newborn and prematures we suggest a cautious regimen in employing this substance, which was until now considered to have no serious side effects.

Arabinonucleotides↗