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At least 145 records · Page 8Linked to original sources
PubMed1994

Centrifugal inputs enhance responses of retinal ganglion cells in the Japanese quail without changing their spatial coding properties.

Centrifugal fibers originating in the midbrain innervate the avian retina. Stimulation of the centrifugal fibers enhances the responses of ganglion cells in the retinas of both chick and pigeon. The enhanced responses have been attributed to disinhibition, a reduction of the inhibitory surround component of the receptive fields of retinal ganglion cells. We found that stimulation of the centrifugal fibers in Japanese quail enhances the responses of retinal ganglion cells to drifting sine-wave gratings over a wide range of spatial frequencies. Our results do not support the idea that centrifugal inputs selectively influence receptive field surrounds. We also found that centrifugal inputs changed the temporal response properties of retinal ganglion cells by enhancing their responses to sine-wave gratings drifting across the retina at higher temporal frequencies (> 5 Hz). The result shows that centrifugal inputs from the midbrain can enhance responses of retinal ganglion cells without affecting the center-surround organization of their receptive fields. The centrifugal modulation of retinal responses may have a role in shifting visual attention.

Animals↗
PubMed1998

Superiority of centrifugal pump over roller pump in paediatric cardiac surgery: prospective randomised trial.

OBJECTIVE: The merits of centrifugal pump in adult cardiopulmonary bypass are well established. This study compares the effects of the Medtronic Biomedicus centrifugal pump with conventionally used roller pump in routine cardiopulmonary bypass in infants and children. METHODS: Between June 1996 and March 1997, 42 children (aged 2 days-13 years) undergoing elective cardiac surgery were assigned to either centrifugal or roller pump bypass. The following variables were studied: haemolysis (haematocrit, free plasma haemoglobin, haptoglobins), platelet activity (platelet counts, Beta-thromboglobulin), leukocyte count, cytokine release (IL-2, IL-6, IL-8), complement activation (C3a and C5a), blood and blood product requirements, urine output on bypass, post-operative blood urea, duration of ventilation, intensive care and hospital stay. RESULTS: Age, weight, disease complexity, duration of bypass, and a number of other variables were comparable in the two groups. The centrifugal pump resulted in lower plasma free haemoglobin (mean +/- SD, 50 +/- 23 vs. 72 +/- 35 mg/dl, P < 0.01), higher platelet count (133.1 +/- 34.8 vs. 63.5 +/- 29.6 x 10(9)/l, P < 0.01), less platelet activation (beta-TG 1253 +/- 633 vs. 1657 +/- 677 ng/ml; P < 0.05), less cytokine release (IL-6 329 +/- 57 vs. 392 +/- 59 pg/ml; P < 0.05), and reduced levels of C3a (4822 +/- 274 vs. 5933 +/- 393 ng/ml, P < 0.01). Differences were detected in favour of the centrifugal pump in urine output on bypass (4.1 +/- 0.5 vs. 2.3 +/- 1.9 ml/kg per h, P < 0.01), post-operative maximal urea (6.5 +/- 3.1 vs. 10.2 +/- 6.7 mmol/l, P < 0.02), ventilation time (18.9 + 6.5 vs. 56.5 + 51.7 h, P < 0.01), duration of intensive care (1.4 +/- 0.79 vs. 3.33 +/- 2.8 days, P < 0.05) and hospital stay (5.7 +/- 1.4 vs. 15.75 +/- 23.9 days, P < 0.01), but not in blood and blood product requirements (RCC: 11.26 +/- 4.6 vs. 10.77 +/- 4.2 ml/kg per 24 h, P > 0.05). CONCLUSION: The centrifugal pump as compared to roller pump results in less blood trauma, reduced platelet activation and less pronounced inflammatory response. There is also an improved renal response during and after bypass. This is translated clinically into reduced requirement for ventilation, shorter intensive care and hospital stays. These results strongly favour the use of centrifugal pump in routine paediatric cardiac surgery.

Adolescent↗
PubMed2005

Preparation of monomodal polyelectrolyte complex nanoparticles of PDADMAC/poly(maleic acid-alt-alpha-methylstyrene) by consecutive centrifugation.

We report on the refinement of anionic and cationic nanoparticles of nonstoichometric polyelectrolyte complexes (PEC) by consecutive centrifugation, which was studied by dynamic light scattering (DLS), atomic force microscopy (AFM), colloid titration and infrared spectroscopy (IR). PEC dispersions were prepared by mixing poly(diallyldimethylammonium chloride) (PDADMAC) and sodium poly(maleic acid-alt-alpha-methylstyrene) (PMA-MS) at the monomolar mixing ratio of n-/n+ = 1.50 (anionic PEC) and 0.66 (cationic PEC), respectively, and the polymer concentration of c(POL) = 0.002 M. The particle size (Rh), titrable charge amount, and IR spectra were determined for both dispersions in the original state, after the first centrifugation and after the second centrifugation. Freshly prepared PEC dispersions contained two different particle sizes: around 10-25 nm (small particles) and around 100 nm (large particles). Consecutive centrifugation of freshly prepared PEC dispersions resulted in the separation of highly charged excess polyelectrolyte (PEL) and small PEC particles from a low charged coacervate phase of the desired larger PEC particles. After the second centrifugation, the coacervate phase of both dispersions PEC-1.50 and PEC-0.66 consisted of monomodal particles sizing around 100 nm. These results were supported by AFM measurements on the respective dispersions deposited on glass plates. PEC-1.50 particles tended to adopt slightly smaller sizes ( approximately 90 nm) in comparison to PEC-0.66 ones (approximately 110 nm). No significant influence of the PDADMAC molecular weight on the particle size was found. IR spectroscopy showed changes in the environment of the carboxylate groups of PMA-MS by consecutive centrifugation. The centrifuged PEC-1.50 dispersions showed remarkable long-term stability over more than a year. The high macroscopic stability of the studied PEC dispersions is presumably due to repulsive electrostatic interparticle interactions and attractive hydrophobic intraparticle interaction. The introduced monomodal PEC particles might be projected as latex analogues or as nanocarriers for drugs and proteins.

Journal Article↗
PubMedDate not supplied

Observations on the removal of microaggregates from stored blood by centrifugation and filtration through a standard 170 micron transfusion filter.

The effect of centrifugation (5 minutes, 6900 X g) on the amount and particle size of blood microaggregates was studied in order to assess whether centrifugation and filtration by a standard (170 micron) transfusion set filter could be used for aggregate removal instead of microfiltration. Aggregates were quantitated using a method based on nylon screens with graded pore sizes. At the beginning of storage, centrifugation increased the particle size of aggregates only slightly. In 7-day-old blood, aggregates capable of passing a 160 micron screen amounted to 308 +/- 107 (SD) mg of debris protein per liter of blood before and 55 +/- 26 (SD) mg of protein (n = 10) after centrifugation. In 14-day-old blood centrifugation increased the size of aggregates even more effectively: 311 +/- 81 (SD) before and 29 +/- 14 (SD) mg of protein following centrifugation. Microaggregates can thus be removed effectively by the centrifugation and filtration method from blood stored for more than 1 week. A standard transfusion set filter can be used instead of a micropore filter.

Blood↗
PubMed1961

Tropic responses of Phycomyces sporangiophores to gravitational and centrifugal stimuli.

A low-speed centrifuge was used to study the tropic responses of Phycomyces sporangiophores in darkness to the stimulus of combined gravitational and centrifugal forces. If this stimulus is constant the response is a relatively slow tropic reaction, which persists for up to 12 hours. The response is accelerated by increasing the magnitude of the gravitational-centrifugal force. A wholly different tropic response, the transient response, is elicited by an abrupt change in the gravitational-centrifugal stimulus. The transient response has a duration of only about 6 min. but is characterized by a high bending speed (about 5 degrees /min.). An analysis of the distribution of the transient response along the growing zone shows that the active phase of the response has a distribution similar to that of the light sensitivity for the light-growth and phototropic responses. Experiments in which sporangiophores are centrifuged in an inert dense fluid indicate that the sensory mechanism of the transient response is closely related to the physical deformation of the growing zone caused by the action of the gravitational-centrifugal force on the sporangiophore as a whole. However, the response to a steady gravitational-centrifugal force is most likely not connected with this deformation, but is probably triggered by the shifting of regions or particles of differing density relative to one another inside the cell.

Fungi↗
PubMed1994

Clinical evaluation of a new type of centrifugal pump.

The major problems with existing centrifugal pumps are leakage, mechanical trauma, and thrombus formation. In consideration of these problems, a new compact centrifugal pump system was developed. The purpose of this study was to evaluate the new centrifugal pump system clinically. Ten patients underwent open heart surgery with a centrifugal pump or a roller pump. During surgery, hemodynamic and hematological data were obtained. A pulsatile assist device in the pump circuit was used in patients with severe heart disease. There was neither operative death nor hospital mortality, and there was no difference with regard to hemodynamic data between the two groups. The centrifugal pump groups, however, had significantly lower hemolysis, especially during prolonged cardiopulmonary bypass. This centrifugal pump could also create sufficient pulsatile flow with a pulsatile assist device. Postoperative macroscopic and microscopic findings demonstrated the smooth surface of the pump without thrombus formation. This centrifugal pump system might be useful for prolonged cardiopulmonary bypass.

Adolescent↗
PubMed1987

[Reorganization of the system of intermediate filaments and detection of the organization centers after centrifugation of cells attached to a substrate].

Cultured pig kidney epithelial cells were centrifuged at 20,000 gav so that the centrifugation force was oriented parallel to the substrate, fixed and processed for indirect immunofluorescent staining with tubulin and vimentin antibodies. After a 2 hour centrifugation vimentin filaments aggregated in the centripetal parts of the cells (probably, because of their association with floating lipid vesicles). Microtubule-organizing centers were found near the centripetal poles of the nuclei, which migrated in the direction of the centrifugal force. The distribution of the cytoplasmic microtubules did not change during centrifugation. The staining of the cultures one hour after centrifugation revealed vimentin-containing spots with radiating intermediate filaments in most of the cells. These spots were localized near the cell nuclei; double immunofluorescent staining with tubulin and vimentin antibodies showed that their position was identical to that of the microtubule-organizing centers. Similar foci of vimentin filaments were seen in the cells after a 3-4 hour centrifugation. Probably, these structures participate in organizing the intermediate filament cytoskeleton in cells.

Animals↗
PubMed1981

Isopycnic-centrifugation studies in caesium chloride and in caesium sulphate on dermatan sulphate proteoglycans from bovine sclera.

1. Two proteodermatan sulphate species from bovine sclera (fractions PG-I and PG-II) separable by gel chromatography were studied by isopycnic centrifugations in CsCl and Cs(2)SO(4) both in an analytical and a preparative mode. 2. In CsCl, fraction PG-I formed a broad band at a density rho=1.75g/ml whereas fraction PG-II banded sharply at rho=1.64g/ml. However, in Cs(2)SO(4), fraction PG-II banded at rho=1.51g/ml and fraction PG-I at rho=1.40g/ml, a reversal of the relative banding positions of the two species in CsCl. 3. Preparative isopycnic centrifugations in the two caesium salts permitted further subfractionation of fractions PG-I and PG-II. In both CsCl and Cs(2)SO(4) fraction PG-I was split into subfractions that varied greatly in uronate/protein ratios but had very similar uronate composition. In contrast, isopycnic centrifugation of fraction PG-II in Cs(2)SO(4) gave rise to subfractions with similar uronate/protein ratios but markedly different uronate composition (iduronate content, 88-42%). 4. Subfractions of fractions PG-I and PG-II obtained in preparative centrifugations in CsCl or Cs(2)SO(4) were examined in the analytical ultracentrifuge. These subfractions banded at discrete positions in the gradient. Estimations of apparent molecular weight for these subfractions from data from the analytical isopycnic centrifugations gave values that were much higher (around 1x10(6)) than were those obtained previously for their ;monomeric' states (fraction PG-I 160000-410000, and fraction PG-II 70000-130000). 5. In CsCl, fraction PG-I may be subfractionated according to the number of side chains in the molecule. Fraction PG-I increased its net solvation (i.e. lowered its buoyant density) to a larger extent than did fraction PG-II in going from CsCl to Cs(2)SO(4) (i.e. from lower to higher water activity). It is proposed that the presence of large amounts of iduronate in fraction PG-II makes these molecules relatively less solvated in Cs(2)SO(4). Thus the uronate composition may be an important factor in determining the banding position of proteodermatan sulphates in density-gradient centrifugations.

Animals↗
PubMed1991

Combining counterflow centrifugal elutriation and glycoprotein Ib-dependent purification of human megakaryocytes: efficacy and selectivity.

To estimate the efficacy of human megakaryocyte purification techniques, mixtures of known numbers of megakaryocytes with a known ploidy range and of bone marrow or peripheral blood mononuclear cells were made. These artificial bone marrow samples were submitted to either a counterflow centrifugal elutriation or Percoll density separation followed by the glycoprotein Ib-dependent agglutination procedure. Also crude bone marrow samples were submitted to counterflow centrifugal elutriation directly followed by a glycoprotein Ib-dependent agglutination. The counterflow centrifugal elutriation resulted in a mean megakaryocyte recovery of 81% (mean 81% +/- 2.3). Purification by glycoprotein Ib-dependent agglutination after either a Percoll density separation or counterflow centrifugal elutriation resulted in a recovery of 61% (mean 61% +/- 15%) and 81% (mean 81% +/- 6) respectively. Purity of the resulting material was 87% (mean 87% +/- 11) and 83% (mean 83% +/- 5) respectively. The various isolation procedures did not affect the ploidy distribution of megakaryocytes greater than or equal to 8N. Counterflow centrifugal elutriation was preferred as the preparing step before glycoprotein Ib-dependent agglutination because of the lower variability in recovery and purity of megakaryocyte populations. When large numbers of rather pure and mature megakaryocytes are required, counterflow centrifugal elutriation followed by the glycoprotein Ib-dependent agglutination is a relatively simple method to purify human megakaryocytes without an appreciable loss in ploidy class greater than or equal to 8N.

Bone Marrow Cells↗
PubMed2000

The use of laboratory centrifugation studies to predict performance of industrial machines: studies of shear-insensitive and shear-sensitive materials.

A method for using a bench-top centrifuge is described in order to mimic the recovery performance of an industrial-scale centrifuge, in this case a continuous-flow disc stack separator. Recovery performance was determined for polyvinyl acetate particles and for biological process streams of yeast cell debris and protein precipitates. Recovery of polyvinyl acetate particles was found to be well predicted for these robust particles. The laboratory centrifugation scale-down technique again predicted the performance of the disc stack centrifuge for the recovery of yeast cell debris particles although there was some suggestion of over-prediction at high levels of debris recovery due to the nature of any cell debris aggregates present. The laboratory centrifuge scale-down technique also proved to be an important investigative probe into the extent of shear-induced breakup of shear-sensitive protein precipitate aggregates during recovery in continuous high speed centrifuges. Such breakup can lead to over 10-fold reduction in separator capacity.

Acceleration↗
PubMed2006

Shear stress analysis of mammalian cell suspensions for prediction of industrial centrifugation and its verification.

This article describes the use of ultra scale-down studies requiring milliliter quantities of process material to study the clarification of mammalian cell culture broths using industrial-scale continuous centrifuges during the manufacture of a monoclonal antibody for therapeutic use. Samples were pretreated in a small high-speed rotating-disc device in order to mimic the effect on the cells of shear stresses in the feed zone of the industrial scale centrifuges. The use of this feed mimic was shown to predict a reduction of the clarification efficiency by significantly reducing the particle size distribution of the mammalian cells. The combined use of the rotating-disc device and a laboratory-scale test tube centrifuge successfully predicted the separation characteristics of industrial-scale, disc stack centrifuges operating with different feed zones. A 70% reduction in flow rate in the industrial-scale centrifuge was shown to arise from shear effects. A predicted 2.5-fold increase in throughput for the same clarification performance, achieved by the change to a centrifuge using a feed zone designed to give gentler acceleration of the bioprocess fluid, was also verified at large-scale.

Animals↗
PubMed1989

Influence of antibiotic impregnation on the fatigue life of Simplex P and Palacos R acrylic bone cements, with and without centrifugation.

The fatigue properties of Simplex P and Palacos R bone cements were compared to their antibiotic impregnated counterparts AKZ* and Palacos R with gentamycin. The effect of porosity reduction by centrifugation of all four cement types was also assessed. Fifteen specimens of each cement type were prepared according to manufacturer's instructions and 15 additional specimens of each cement type were prepared by mixing the powder with chilled monomer (0 degrees C) and then centrifuging the cement immediately after mixing. Fifteen fully reversed tension-compression fatigue tests were performed at 15 MPa in stress control for each cement preparation in vitro while simulating the in vivo state (37 degrees C and 100% humidity). The number of cycles to failure were recorded. There was no significant difference in the fatigue life of Palacos R and Simplex P when both cements were prepared in the standard fashion. The addition of 1/2 g of gentamycin to Palacos R did not significantly alter its fatigue properties. The addition of 0.5 g of erythromycin and 0.24 g of colistin did not decrease the fatigue life of Simplex P. Centrifugation significantly improved the fatigue properties of Simplex P and AKZ. The fatigue lives of Palacos R and Palacos R with gentamycin were not improved by centrifugation. The fatigue life of centrifuged Simplex P was significantly greater than the fatigue life of Palacos R and of Palacos R with gentamycin, whether the Palacos R based cements were centrifuged or not.

Biocompatible Materials↗
PubMed1998

Effects of centrifugation and lipid removal on the cryopreservation of in vitro produced bovine embryos at the eight-cell stage.

The effects of intracellular lipid polarization and lipid removal treatments on the postthawed in vitro development of frozen bovine embryos at the 8-cell stage were studied. As the first step, bovine presumptive zygotes were centrifuged at 16,000 g for 20 min for the cytoplasmic lipid polarization and their lipid layers were removed by micromanipulation in order to examine the influence of these treatments on the developmental capacity of bovine zygotes. As the second step, bovine embryos developed to the 8-cell stage following centrifugation treatment at various forces (8000, 12,000, and 16,000 g) or lipid removal treatment at the 1-cell stage were frozen in 1.8 M ethylene glycol + 0.05 M trehalose supplemented with 5% polyvinylpyrrolidone in a one-step procedure. There were no significant differences among the control (nontreatment), lipid-polarized, and lipid-removed groups with respect to the developmental capacity of fresh nonfrozen zygotes (experiment 1). The rates of survival and development to the blastocyst of frozen-thawed 8-cell embryos increased slightly with increasing force of centrifugation (experiment 2). The rate of development into blastocysts of the frozen-thawed 8-cell embryos was significantly higher in the groups that underwent centrifugation (at 16,000 g for 20 min; P < 0.05) or lipid removal (P < 0.01) treatments than the control (intact) group. However, there were no significant differences among the groups with respect to the rate of development to the expanded/hatched blastocyst stage. In addition, the mean cell numbers of embryos developed into blastocysts (day 8) derived from frozen-thawed 8-cell embryos tended to be low in the centrifugation and lipid removal groups compared to the controls (experiment 3). These results suggest that although the centrifugation with or without lipid removal treatments has no detrimental effects on the developmental capacity of bovine zygotes, the freezing tolerance of bovine 8-cell embryos was not improved by these treatments.

Animals↗
PubMed1977

Effect of chronic centrifugation on bone density of the rat.

Alterations in density of the femur of female Sprague-Dawley rats exposed to chronic centrifugation of 2.76 G for 810 days were studied. The density was measured by photon absorptiometry, using a 125I Profile Scanner. The photon absorption measurements were taken: (1) on bone sections from the midshaft of the femur in the anterior, posterior, medial and lateral cortex with the beam path parallel to the shaft axis, and (2) on the femur near the midshaft with the beam path perpendicular to the shaft axis. The latter were correlated with the cross-sectional area in the midshaft. Compared with femora of age-matched rats (age controls) and of rats having body masses comparable to the centrifuged group (weight controls), kept under earth gravity, the chronically centrifuged animals showed no average no significant differences in photon absorption (density) in the four positons in midshaft. The absorption measurements perpendicular to the shaft axis in all groups differ significant differences between the centrifuged group and the control groups. When cross-sections of equal size are compared, the density of the rat femora is clearly higher in the centrifuged animals than in the control groups. In contrast, the age and weight controls do not differ significantly in density. The results suggest that the increased stress in the rat femora due to centrifugation is reflected in an increased bone density.

Absorption↗
PubMed1990

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge.

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100-500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10(7) cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.

Animals↗
PubMed1977

A new type of cytocentrifuge, the valve-centrifuge.

A new type of cytocentrifuge has been developed in which the sedimentation process of the cells onto the slides is separated from the draining of the sedimentation fluid. This is realised by electrically controlled valves which can be closed and opened while the centrifuge is running. Sedimentation is carried out with closed valves, draining of adhering medium with open valves. The preparations, freed of adhering medium by the centrifugal force can be taken out and the cells can be fixed. Alternatively the valves can be closed again and fixative can be introduced through a central well, the cells still being under the influence of the centrifugal force. With subsequent draining of the fixative and introduction of washing and staining solutions through the central well, the whole process from sedimentation to staining can be carried out in the running centrifuge. The process seems well suited for complete automation. Using dilution series from a suspension of human buffy coat cells counted in a Buerker chamber, the cell counts in the centrifuge preparations showed virtually total recovery of cells, with no apparent selection or specific distribution of cell types. Draining of the sedimentation and fixative fluids at a slow rate was found to be vital for optimal recovery of cells. The morphology of different cell types sedimented on the slide was excellent. The flattening of nuclei thorugh gravity was studied by cytophotometry of Feulgen-stained leucocytes. The nuclear area of these cells was found to be approximately double that from cells in identically stained classical smears. With this type of valve-centrifuge a quantitative and unbiased recovery of uniformly spread and flattened cells on coverslips or slides may be obtained, thus making the procedure well suited to automated analysis based on cytophotometric principles and morphometric pattern recognition.

Animals↗
PubMed1993

Centrifugation causes adaptation of microfilaments: studies on the transport of statoliths in gravity sensing Chara rhizoids.

The actin cytoskeleton is involved in the positioning of statoliths in tip growing Chara rhizoids. The balance between the acropetally acting gravity force and the basipetally acting net outcome of cytoskeletal force results in the dynamically stable position of the statoliths 10-30 micrometers above the cell tip. A change of the direction and/or the amount of one of these forces in a vertically growing rhizoid results in a dislocation of statoliths. Centrifugation was used as a tool to study the characteristics of the interaction between statoliths and microfilaments (MFs). Acropetal and basipetal accelerations up to 6.5 g were applied with the newly constructed slow-rotating-centrifuge-microscope (NIZEMI). Higher accelerations were applied by means of a conventional centrifuge, namely acropetally 10-200 g and basipetally 10-70 g. During acropetal accelerations (1.4-6g), statoliths were displaced to a new stable position nearer to the cell vertex (12-6.5 micrometers distance to the apical cell wall, respectively), but they did not sediment on the apical cell wall. The original position of the statoliths was reestablished within 30 s after centrifugation. Sedimentation of statoliths and reduction of the growth rates of the rhizoids were observed during acropetal accelerations higher than 50 g. When not only the amount but also the direction of the acceleration were changed in comparison to the natural condition, i.e., during basipetal accelerations (1.0-6.5 g), statoliths were displaced into the subapical zone (up to 90 micrometers distance to the apical cell wall); after 15-20 min the retransport of statoliths to the apex against the direction of acceleration started. Finally, the natural position in the tip was reestablished against the direction of continuous centrifugation. Retransport was observed during accelerations up to 70 g. Under the 1 g condition that followed the retransported statoliths showed an up to 5-fold increase in sedimentation time onto the lateral cell wall when placed horizontally. During basipetal centrifugations > or = 70 g all statoliths entered the basal vacuolar part of the rhizoid where they were cotransported in the streaming cytoplasm. It is concluded that the MF system is able to adapt to higher mass accelerations and that the MF system of the polarly growing rhizoid is polarly organized.

Acceleration↗
PubMed1995

Morphological and electrophysiological properties of centrifuged stratified Xenopus oocytes.

To separate and concentrate various cytoplasmic organelles in wild type and albino Xenopus oocytes, defolliculated cells were loaded on a Ficoll-400 gradient and centrifuged. Optimum results were obtained with centrifugations at 10,000 g for 5 min at 20 degrees C. The cells became pear-shaped and appeared stratified with the white lipid yolk on top, an intermediate transparent zone of about 100-300 microns, and the greenish protein yolk at the bottom. To determine the cellular constituents, particularly of the transparent zone, electron microscopy was performed. The transparent zone was found to contain (from animal to vegetal) the various endoplasmic reticula, a layer of mitochondria, cytoplasm enriched in ribosomes and the depressed nucleus. In centrifuged stratified wild type oocytes, most of the pigment was layered on top of the protein yolk. The typical cortical aspects of the oocyte persisted. Centrifuged albino oocytes had a very pronounced transparent zone with sharp transitions to the lipid phase and to the protein yolk. The resting membrane potentials of centrifuged oocytes were between -35 and -65 mV, and the membrane resistances were in the 500 k omega to 1 M omega range. Under voltage clamp conditions, the oocytes exhibited Ca(2+)-activated Cl- currents with biphasic kinetics and spontaneous oscillations of these currents. It is concluded that centrifuged stratified oocytes have normal electrophysiological properties, and that they are a suitable preparation to study the contribution of various cellular organelles to the propagation of second messengers in the cytosol.

Animals↗