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Prolonged extracorporeal membrane oxygenation in sheep with a hollow-fiber oxygenator and a centrifugal pump.

ECMO with a roller-pump employs two potentially thrombogenic devices: the servoregulator bladder and heat exchanger. To eliminate these we used an 0.8 m2 Capiox II hollow-fiber oxygenator ventilated with warmed humidified oxygen and a Bio-Medicus centrifugal pump with a 1/4 inch head at a flow of 250 cc/min in six sheep during 96 hr of ECMO each. Oxygenator performance and plasma Hgb were determined, as were the volume, electrolyte, and protein content of fluid accumulating in the gas phase of the oxygenator. Mean oxygen transfer was 15.78 +/- 3.15 ml/min, and mean differences in PaO2 between blood entering and leaving the oxygenator was 360 +/- 49 mmHg. Mean plasma Hgb was 24.7 +/- 12.8 mg/dl. Fluid in the gas phase of the oxygenator was less than 75 ml/day and contained no albumin, protein, sodium, potassium, or chloride. Centrifugal pumps have been associated with hemolysis at low flows, but this may have been due to the simultaneous use of silastic membrane oxygenators that have high resistance. Hollow-fiber oxygenators have been associated with early failure of gas exchange and fluid accumulation in the gas phase. This may have been due to ventilation with dry gas. We conclude that a hollow-fiber oxygenator and a centrifugal pump can provide excellent gas exchange, acceptable hemolysis, and little fluid loss.

Animals↗

Centrifugal organization of direction preferences in the cat's lateral suprasylvian visual cortex and its relation to flow field processing.

The cerebral cortex of the cat contains between 1 and 2 dozen representations of the visual field with different functional specializations. Six visual field maps lie along both banks of the suprasylvian sulcus, lateral and anterior to the visual areas in the occipital cortex. We have studied single-unit receptive field properties and their global organization across the visual field in 2 of these lateral suprasylvian areas, PMLS (essentially the Clare-Bishop area) and PLLS. Most neurons in PMLS and PLLS display selectivity for the direction of a light stimulus moving across their receptive fields with various degrees of directional tuning. We have used light spots of different size and velocity projected on a tangent screen in order to determine the direction preference of cells in these 2 areas. A strong tendency was found for neurons to respond best to centrifugal directions, i.e., to movement away from the area centralis. Thus, for these cells direction preference depends on the location of their receptive fields within the visual field. Velocity preference and binocular interaction in these neurons is also globally organized: Velocity preference increases with eccentricity, binocular synergism is strongest in the center of the visual field. Cluster analysis of recording tracks with respect to "radial" and "circular" cell categories reveals a grouping of cells with like properties in the lateral suprasylvian cortex. These new categories are formed by combining "centrifugal" and "centripetal" cells on the one hand and cells with direction preferences orthogonal to these on the other. The radial or centrifugal organization of direction preferences in conjunction with the global arrangement of velocity preference and binocular interaction suggests that PMLS and PLLS are involved in the processing of expanding visual flow fields of motion. Such flow fields are commonly encountered when a visual object moves towards an observer or during forward locomotion.

Action Potentials↗

Studies on peroxisomes. V. Effect of ethyl p-chlorophenoxyisobutyrate on the centrifugal behavior of rat liver peroxisomes.

After Wistar male rats had been fed on a diet containing 0.25% of ethyl p-chlorophenoxyisobutyrate (CPIB) for 28 days, changes in the enzyme activities and centrifugal behavior of rat liver peroxisomes were investigated. (1) Compared with control rats fed on the basal diet, the catalase [EC 1.11.1.6] activity of rat livers after the administration of CPIB increased about 2.5-fold, while urate oxidase [EC 1.7.3.3] activity did not change significantly. Though D-amino acid oxidase [EC 1.4.3.3] activity markedly decreased to approximately one-sixth of the control, the activity of L-alpha-hydroxy acid oxidase [EC 1.1.3.15], a flavin enzyme like D-amino acid oxidase, was not affected significnatly after the administration of CPIB. (2) When the hepatic cells of CPIB-treated rats were fractionated by differential centrifugation, most of the increase of catalase activity appeared in the supernatant fraction. A decrease in the hepatic D-amino acid oxidase activity of CPIB-treated rats was observed in all the fractions. As for the subcellular distribution of the particle-bound enzymes, the specific activities of both catalase and urate oxidase of CPIB-treated rat livers were higher in the light mitochondrial fraction than in other fractions. (3) Sedimentation patterns in a sucrose density gradient did not show any difference between normal peroxisomers, and CPIB-treated ones. (4) In the case of CPIB-treated rats, studies of their sedimentation patterns by Ficoll density gradient centrifugation showed two main particulate peaks containing both catalase and urate oxidase, although only a single peak was observed in the case of control rats.

Alcohol Oxidoreductases↗

Bone remodeling in centrifuged rats: histomorphometric study after an 18-day run.

A histomorphometric and histodynamic study of bone tissue was conducted in rats exposed to 2 G centrifugation for a period of 18 d. Shortening of the femurs was observed, associated with alterations of the growth cartilage in centrifuged animals. The morphometric analysis demonstrates a reduction in bone volume without increase in the activity of osteoclasts. The fluorescent fixation during the dynamic study shows a reduced appositional rate. This result seems to reflect the numerous factors which contribute to the experimental centrifugation test: the osteoporosis is cortisone induced; it is a response to the aggression and probably conceals the remodeling due to higher stress loads.

Animals↗

[Functional state of the cardiovascular system system during combined exposure to 28-day immersion, rotation in a short radius centrifuge, and physical loading on a bicycle ergometer].

The cardiovascular function of four test subjects exposed to 28-day "dry" immersion was examined before and after 6-day cycles of rotation in a short-arm centrifuge to provide 1-2 Gz, bicycle ergometer exercise, and their combination. An exposure to acceleration of 3 Gz in a 7.25 m arm centrifuge was used as a provocative test. The above countermeasures reduced but not eliminated entirely immersion-induced cardiovascular deconditioning. From this study a combined use of acceleration of 1-2 Gz in a short-arm centrifuge and bicycle ergometer exercise can be recommended as a countermeasure against cardiovascular deconditioning in weightlessness.

Blood Pressure↗

Comparison of turbidimetric and light-scattering measurements of immunoglobulins by use of a centrifugal analyzer with absorbance and fluorescence/light-scattering optics.

We have developed a centrifugal analyzer with both fluorescence/light-scatter and conventional absorbance optics. The instrument is used in this investigation to study the formation of antigen-antibody complexes by light scattering and turbidimetric measurements, and to develop assays for human immunoglobulins G, A, and M. Concentrations are calculated from a nonlinear least-squares fit of calibrators, and antigen excess is automatically detected from kinetic curve characteristics. Precisions and patients' results are presented, and assay sensitivity and reliability in the detection of antigen excess are compared. We also investigated the effects of centrifugal force on complex formation. Both nephelometry and turbidimetry can be very satisfactorily adapted to centrifugal analyzers. We present a model to describe the observed differences between the light-scatter and the turbidity data.

Antigen-Antibody Complex↗

[Clinical experience of extracorporeal life support with the use of centrifugal pump ECMO].

Many reports have documented the good results with the use of extracorporeal life support in severe respiratory and circulatory failure. In our institution, we have used the centrifugal pump (Bioconsol 540: Biomedicus Co. Ltd.) ECMO for extracorporeal life support, and obtained good results in three cases. We used V-V bypass ECMO in one case (idiopathic interstitial pneumonia) and V-A by-pass ECMO in other two cases (ARDS and meconium aspiration syndrome). Duration of total bypass in three cases were 34, 51 and 140 hours, respectively. Recent reports on the use of centrifugal pump have indicated that it can bring excellent results of circulatory assist for cardiogenic shock. Compared to the roller pump method, the use of centrifugal pump ECMO was very simple, and could serve as an effective method for providing improvement in a relatively short period.

Adult↗

[Clinical experience of VA-bypass as a cardiac assist system using centrifugal pump with membranous oxygenator in patients with profound ventricular failure after cardiac surgery].

Twelve patients including 6 ischemic heart diseases, 5 valvular diseases, and one adult atrial septal defect combined with mitral and tricuspid valve insufficiency were treated with circulatory assist system for postoperative severe low-cardiac output syndrome. Intra-aortic balloon pumping (IABP) was used in all cases. In early 7 cases, left heart bypass system was employed including one roller pump, one left ventricular assist device (LVAD), and 5 centrifugal pump. Only one LVAD case could be weaned but died of sustained ventricular arrhythmias 5 days postoperatively. In recent 5 cases, veno-arterial bypass (VA-bypass) was employed using centrifugal pump with membranous oxygenator. Venous blood was drained from the right atrium and oxygenated blood was returned to the right axillary artery. Real time reinfusion of drained blood from intrapericardial and mediastinal tube to the central vein served efficiently to maintain hemodynamics and to save homologous blood transfusion. Three of 5 cases who were treated with Va-bypass survived and are doing well. We conclude that VA-bypass consisting with centrifugal pump and oxygenator returning oxygenated blood to the right axillary artery combined with IABP were useful strategies as biventricular support for profound cardiac failure following surgery.

Aged↗

Stimulatory effect of counterflow centrifugal elutriation in large-scale separation of peripheral blood monocytes can be reversed by storing the cells at 37 degrees C.

Transfusion of peripheral blood monocytes may be of benefit as adjuvant treatment of leukopenic patients with serious infections. To study the feasibility of this approach, a large-scale monocyte separation procedure employing leukapheresis, density gradient centrifugation, and counterflow centrifugal elutriation was established. By processing 5 to 6 liters of normal donor blood, it was possible to obtain a mean of 1.1 x 10(9) (range: 0.5-1.7 x 10(9) cells) of mononuclear cells, of which 89% (range: 82-94%) were monocytes by Wright's stain morphology. When the elutriation was performed in XVIVO-10, a commercially available, serum-free medium developed for adoptive immunotherapy, spontaneous secretion of superoxide by the monocytes was significantly higher than for monocytes elutriated in Hanks' balanced salt solution without calcium and magnesium or non-elutriated peripheral blood mononuclear cells. This stimulated state of the monocytes was observed both immediately after elutriation and after overnight storage at 4 degrees C, and it was not affected by the type of storage vessel used. Overnight storage of the monocytes at 37 degrees C resulted in a reversal of the stimulated state of the cells. Monocytes elutriated in XVIVO-10 and kept overnight at 4 degrees C released high amounts of arachidonic acid. A subsequent decrease in this release was seen after additional storage at 37 degrees C for 18 hours. These observations demonstrate that separation and storage variables have important effects on the state of stimulation of monocytes. Further investigations of such variables may suggest improved procedures for preparation and storage of these cells, as well as possible ways to stimulate monocytes prior to transfusion.

Arachidonic Acid↗

Improved isolation and purification of rat liver peroxisomes by combined rate zonal and equilibrium density centrifugation.

Two different peroxisome preparations were isolated from male rat liver by using total homogenate (TH) as the starting material for one and the light mitochondrial (L) fraction for the other. The technique worked out is based on rate zonal (RZ) centrifugation in a sucrose gradient and subsequent isopycnic centrifugation in a Nycodenz gradient. The peroxisome fraction isolated from the L fraction consisted of 97-98% peroxisomal protein with catalase activity 49-fold enriched over TH. The peroxisome preparation isolated directly from TH represented about 55% of the total liver peroxisome population and had catalase activity 43-fold enriched compared with TH. The contribution of peroxisome protein to the liver protein was calculated to be in the range 1.82-2.02%. Peroxisomes isolated from TH were considerably more heterogeneous in size than peroxisomes isolated from the L fraction. Comparison of the polypeptide patterns of both preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed some quantitative differences. Several major polypeptides were found to be exclusively located in the peroxisome membrane. These polypeptides migrated in the gel with apparent molecular masses of 69, 42.5, 36, 26, 21, and 15 kDa.

Animals↗

Centrifugation equilibrium for spheres and spherocylinders.

The centrifugation equilibrium problem is formulated and solved using a new procedure in which the specified variables are the temperature, system volume, particle dimensions and concentrations, angular speed, cell length, and cell distance from the rotation axis. As a result, we obtain the concentration profiles for all types of particles present in the system, which are considered to be immersed in a fluid. The particles are modeled as hard nonattractive spherocylinders using an equation of state, but the procedure is not restricted to any geometrical shape, and can be used with any equation of state available. The fluid is treated as a continuous medium, responsible for centrifugal buoyancy. We make calculations for colloidal suspensions of silica, often used for separations in biotechnology. Results are in good agreement with experiments and show excellent agreement in comparison with Monte Carlo simulations. Our calculations also predict focusing and shifting phenomena that have been experimentally observed in separations of fine particles.

Centrifugation↗

Macro and micro rate zonal analytical centrifugation of polydisperse and slowly diffusing sedimenting systems in isovolumetric density gradients. Application to cartilage proteoglycans.

A method to study the polydispersity of zonally sedimenting and slowly diffusing macromolecules or particles in isokinetic or isovolumetric density gradients is presented. First, a brief theory is given for predicting the zonal profile after a "triangular" (or "inverse") zone is centrifuged. This type of zone is essential to preserve hydrodynamic stability of the very slowly diffusing polydisperse solutes. It is proven, both by semitheoretical considerations and by computer calculations, that the resulting concentration profile of macrosolute is almost identical with that obtainable with a rectangular zone coextensive with the triangular one and carrying the same total mass. Next, practical procedures are described for the convectionless layering of very small triangular zones (50 microL or less). The linearity and stability of the zones are experimentally tested and verified. Finally, the method is applied to cartilage proteoglycan preparations that included either the monomeric molecules only or both the monomeric and the aggregated ones. The zonal results are compared with those obtained by using conventional boundary sedimentation. The two sets of results are seen to coincide fairly well, thus proving that the present technique can add to preparative zonal centrifugation the analytical precision of boundary sedimentation. A multimodal polydisperse system is suggested to describe the aggregated proteoglycan macromolecules.

Animals↗

Subcellular fractionation by differential and zonal centrifugation of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis.

1. Homogenates were prepared from sphaeroplasts of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis and the distributions of marker enzymes were investigated after differential centrifugation. Cytochrome c oxidase and cytochrome c were sedimented almost completely at 10(5)g-min, and this fraction also contained 37% of the catalase, 27% of the acid p-nitrophenyl phosphatase, 53 and 54% respectively of the NADH- and NADPH-cytochrome c oxidoreductases. 2. Zonal centrifugation indicated complex density distributions of the sedimentable portions of these enzymes and of adenosine triphosphatases and suggested the presence of two mitochondrial populations, as well as a bimodal distribution of peroxisomes and heterogeneity of the acid p-nitrophenyl phosphatase-containing particles. 3. Several different adenosine triphosphatases were distinguished in a post-mitochondrial supernatant that contained no mitochondrial fragments; these enzymes varied in their sensitivities to oligomycin and ouabain and their distributions were different from those of pyrophosphatase, adenosine phosphatase and adenosine pyrophosphatase. 4. The distribution of NADPH-cytochrome c oxidoreductase demonstrated that it cannot be used in S. carlsbergensis as a specific marker enzyme for the microsomal fraction. Glucose 6-phosphatase, inosine pyrophosphatase, cytochrome P-450 and five other enzymes frequently assigned to microsomal fractions of mammalian origin were not detected in yeast under these growth conditions.

Adenosine Triphosphatases↗

The resolution of mixtures of viable mammalian cells into homogeneous fractions by zonal centrifugation.

Large-scale separation of mixtures of mammalian cells was obtained with the A-1X zonal centrifuge rotor and density gradients consisting of Ficoll dissolved in modified Eagle's MEM suspension-culture medium. The cells remained viable as tested by plating efficiency or by motility observed with time-lapse photography. Rabbit thymocyte and HeLa cell mixtures were separated with 99 and 89 per cent purity, respectively. Mixtures of thymocytes and suspension-cultured, human acute leukemia cells (Roswell Park strain LKID) were separated with 93 and 91% purity, respectively. HeLa cells were isolated 92% pure from a mixture with horse leukocytes. A book of charts giving the sedimentation position and velocity versus time of cells in the A rotor under standard conditions of gradient composition, angular velocity, and temperature was prepared with the use of a computer program based on the differential sedimentation equation. The charts are used to estimate the centrifugation time necessary for maximum separation of cells. The success achieved in separating mixtures of cells points to the future possibility of large-scale fractionation of solid tissues, especially tumor tissues, into preparations cf viable cells of a single type.

Animals↗

Fractionation by differential and zonal centrifugation of spheroplasts prepared from a glucose-repressed fission yeast Schizosaccharomyces pombe 972h-.

A method is described for the preparation of spheroplasts in high yield from Schizosaccharomyces pombe, by treating cells grown in the presence of glucose and deoxyglucose with snail digestive enzymes. Gentle disruption of such spheroplasts yielded homogenates, from which marker enzymes for nuclei (NAD pyrophosphorylase) and mitochondria (cytochrome c oxidase activity and spectroscopically-detectable cytochromes a + a3) could be quantitatively sedimented by low-speed centrifugation. In contrast to previous findings with Saccharomyces carlsbergensis, cytochrome c oxidase and another mitochondrial enzyme, succinate dehydrogenase, were completely sedimentable by zonal centrifugation in sucrose gradients in the presence of either 2 mM-MgCl2 or 0-4 mM-EDTA. Mitochondria were apparently smaller and of lower buoyant density in gradients containing EDTA. The bulk of the total units of malate dehydrogenase and NADH; cytochrome c oxidoreductase sedimented with mitochondria, whereas NADPH: cytochrome c oxidoreductase was located in fractions containing no mitochondria. The distributions of mitochondrial enzymes were heterogeneous in populations of mitochondria separated on the basis of size or density. The possible origins of mitochondrial heterogeneity in extracts of S. pombe are discussed with special reference to changes in the enzyme activities of cells during the cell cycle.

4-Nitrophenylphosphatase↗

Counterflow centrifugation of red cell populations: a cell age related separation technique.

Red cell populations were fractionated on the basis of differences in density by a centrifugation method described by Murphy and on the basis of differences in mean cell volume (MCV) by counterflow centrifugation. By 59Fe-incorporation and determination of the HbA1c content, both methods were studied for their ability to separate red cell populations into fractions of different mean cell age. It can be concluded that separation on the basis of differences in cell volume results in a linear separation according to age whereas separation on the basis of density only results in an accumulation of very young cells in the top fractions. A gradual decrease in cell volume with age, combined with a constant haemoglobin concentration in the cells, indicates release of haemoglobin from the red cells during their lifespan.

Cell Separation↗

Separation of Treponema pallidum from tissue substances by continuous-flow zonal centrifugation.

The zonal ultracentrifuge was used for separation of Treponema pallidum from large volumes of rabbit testicular syphiloma extracts by continuous-flow centrifugation in a cesium chloride density gradient. The gradient was linear with radius from a density of 1.05 to 1.36 g/ml. Operating speeds were 15,000 rev/min for the continuous-flow phase and 25,000 rev/min for a 30-min banding period. A total of 9 x 10(9) (24.3%) treponemes were recovered from the original extract. Of the treponemes recovered, 88% formed a band at a density of 1.170 to 1.211 g/ml. Within the limits of present methods of assay, these fractions were relatively free from testicular particulates and protein when compared with treponemes recovered after differential centrifugation. Observations of the isolated fractions by dark-field and electron microscopy indicated a lack of gross morphological damage to T. pallidum. Their antigenic characteristics were also retained, as evidenced by their ability to react with syphilitic sera in the indirect fluorescent-antibody procedure.

Animals↗

Comparison of the effects of filtration leucapheresis and discontinuous flow centrifugation leucapheresis on granulocyte microbicidal function.

In an investigation of the in vitro phagocytic and microbicidal function of granulocytes collected by filtration leucapheresis (FL) from 18 donors and by discontinuous flow centrifugation leucapheresis (DFC) from six donors, comparison was made with the function of granulocytes obtained from the same donors by venepuncture and density gradient centrifugation over Ficoll-Isopaque (FI). No significant impairment of the phagocytosis or killing of Candida guilliermondii by either FL- or DFC-granulocytes was observed. Although the ability of FL-granulocytes to phagocytose and kill Staphylococcus aureus did not differ significantly from the function of control FI-granulocytes, DFC-granulocytes were significantly less active.

Adult↗