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Alzheimer's disease: distribution of protein on sucrose density gradient centrifugation.

Material has been examined from eighteen brains, postmortem and from 3 cortical biopsies. The case were classified on clinical and morphological criteria and include cases of dementia and of nonneurological disease. Fractions were isolated from homogenates of neocortex and caudate nucleus by means of discontinuous sucorse density gradient centrifugation. The fractions were analysed for protein content. The mean protein content of fraction 1, at the interphase between 0.32 M and 0.8 M sucrose, from the cortex of the 6 Alzheimer cases was reduced. (P less than 0.005) by 25%. This deficit in protein content represents less than 5% of the total tissue protein. There were no significant differences in the protein content between other fractions from the control and Alzheimer cases.

Adult↗

Preparative density gradient centrifugation of RNA and DNA in cesium sulfate-urea mixture.

A new procedure is described for a large scale separation and purification of unfixed DNA and RNA from a mixture of partially extracted nucleic acids and lysates of subcellular fractions by centrifugation to equilibrium in cesium sulfate-urea mixture. Optimum conditions are described for the separation and quantative recovery of both RNA and DNA in a pure form. The procedure allows determination of peak buoyant densities of 4-5s RNA, 7-11s mRNA and total cytoplasmic RNA. The procedure also allows fractionation of small molecular weight classes of cytoplasmic RNAs from the 18s and 28s rRNAs.

Animals↗

Purification of metacyclic forms of Trypanosoma cruzi by Percoll discontinuous gradient centrifugation.

A method for the purification of metacyclic forms of Trypanosoma cruzi has been developed. Metacyclic forms obtained in modified Grace medium were separated from the epimastigote forms by Percoll discontinuous density gradient centrifugation. Four different osmotic pressures were applied: 160 +/- 10, 260 +/- 10, 310 +/- 10 and 510 +/- 10 mosmol/kg H2O. At 160 mosmol/kg H2O, 100% of the metacyclic forms with a 21.3% yield were found in the interphase 1.120/1.125 g/ml, while 92.7% of the metacyclic forms with a 73.7% yield were found in the interphase 1.115/1.120 g/ml. At 310 mosmol/kg H2O, 100% of the metacyclic forms in the interphase 1.135/1.140 g/ml with a 36.8% yield were obtained. Metacyclic forms purified in this way do not show alterations in their capacity to infect cultures of HeLa cells.

Animals↗

Fractionation of Pneumocystis carinii developmental stages by counterflow centrifugal elutriation and sequential filtrations.

Immunomagnetic sorting, sequential filtrations, and counterflow centrifugal elutriation were compared for their ability to obtain enriched populations of Pneumocystis carinii developmental stages from infected rat-lung homogenates. Elutriation combined with sequential filtrations resulted in highly (> 95%) enriched populations of P. carinii cysts and trophozoites with excellent viability. This approach offers advantages over previously described methods of obtaining enriched P. carinii cell populations and should have important applications to research on this organism.

Animals↗

[Harvesting of granulocytes for granulocyte transfusion by means of continuous flow centrifugation or filtration leukapheresis].

More than 10(10) viable granulocytes are necessary for a therapeutical effective granulocyte transfusion. This number of cells can be harvested from normal donors by two techniques basing on different principles: continuous flow centrifugation (CFC) and filtration leucapheresis (FL). Our studies demonstrated that, under certain special conditions, the separation potentials of both methods are comparable yielding 2.5 to 3.0 X 10(10) granulocytes within 4 hrs. Granulocyte collection rate was optimal if donors were treated with dexamethasone during 16 hrs prior to the state of the procedure. However, the costs of CFC exceed those of FL by a factor of about two. The increased occurrence of side effects attributed to the transfusion of FL-granulocytes can be reduced to the level of CFC-granulocytes by repetitive filtration-elution leucapheresis minimizing cell damage. The studies define the efficiency spectrum of CFC which in addition to granulocyte separation includes collection of thrombocytes, cells for immunotherapy, and plasmapheresis.

Blood Transfusion↗

The utilization of a new strength citrate anticoagulant during centrifugal plateletpheresis. II. Assessment of in vitro platelet function.

Platelets contained in concentrates prepared by intermittent flow centrifugation utilizing 5.0 g total ionized citrate, function normally as assessed by tests for adhesion, spreading, and ADP and collagen-induced aggregation. The ability of platelets to adhere and to aggregate following incubation with ADP is significantly reduced, in samples taken immediately after the collection procedure. Total platelet collection and platelet collection efficiency, is not compromised by use of the above citrate concentration.

Anticoagulants↗

Preparation of peroxisomes from carp liver by zonal rotor density gradient centrifugation.

Peroxisomes from carp liver can be separated by isopycnic density gradient centrifugation in sucrose. Without reaching complete sedimentation equilibrium, the purification by this method is quite successful. There is a 40-fold enrichment of catalase, the peroxisomal marker, with a total yield of 27%. No pretreatment of animals is necessary for separation from lysosomes, which, besides high fragility, show lower buoyant densities than peroxisomes. The enzyme content of carp liver peroxisomes is similar to that of rat liver, with the exception of alpha-glycerophosphate dehydrogenase, which in this tissue is a completely soluble cytoplasmic enzyme. Total activities are much lower than in the rat, for the characteristic peroxisomal oxidases the difference being in the range of one order of magnitude.

Alcohol Oxidoreductases↗

Variation in monoamine oxidase activity in rat brain crude mitochondrial fractions prepared by rate zonal centrifugation.

Rat brain crude mitochondrial fractionated by rate zonal centrifugation using an iso-osmotic gradient of Ficoll and sucrose. The results demonstrated that the isolated fractions were biochemically heterogeneous with regard to the enzymes, monoamine oxidase (MAO), NADH dehydrogenase and succinate dehydrogenase. When the activity of MAO was plotted as % of the highest specific activity towards tyramine, kynuramine oxidation remained fairly constant in fractions 10 to 30 but tyramine and dopamine showed separate peaks of activity in fractions 21 and 32 respectively. Sonic oscillations of separated particulate fractions did not change the ratios of various monoamine deamination when compared to the intact particles.

Animals↗

Wall effects in sucrose density gradient centrifugation of viruses.

The depletion of sedimenting particles to be expected from wall effects in the course of sucrose gradient centrifugation, was computed for various types of swing-out rotors on the assumption that particles hitting the tube wall are lost from the gradient. This assumption was roughly verified for heat-denatured, but not for native poliovirus. Tween 20 reduced the depletion, and prevented tight sticking of heat-denatured virus particles to tube walls.

Capsid↗

Clarification of animal cell cultures on a large scale by continuous centrifugation.

Animal cells from 80-L and 2000-L fed batch fermentations were removed by a prototype disc stack centrifuge in order to achieve a fast and reliable separation of solids from large quantities of cell culture fluids. The clarification capacity was excellent for animal cells but particles remained in the liquid phase and affected further downstream processing of the cell-free harvest fluid. No significant loss of product was observed. A number of parameters were monitored to optimize process conditions for use with animal cells.

Animals↗

[In-vitro formation of plasma polyps in the human placenta and their isolation by means of "inverted centrifugation" (author's transl)].

1. Histological observations on sliced human placenta shaked in Krebs-Ringer-solution showed an high plasma polyp formation by the syncytiotrophoblast without any monoidoacetate or sodiumfluoride poisoning [1]. 2. The high plasma polyp formation of placenta slices incubated with gased (96% O2, 4% CO2) Krebs-Ringer-solution and shaked at 37 degrees C decreased rapidly and lasted up to 40 min. 3. Perfusion of the placenta intervillosum with fetal serum added Krebs-Ringer-Solution gave results in plasma polyp production up to six times higher than serum free Krebs-Ringer-solution perfusions. 4. Plasma polyp isolation and concentration by one step is discribed applying the inverted centrifugation technique: Plasma polyps migrate centripetally in Krebs-Ringer-solution containing 6% Ficoll (Pharmacia) and were concentrated in the tip of a tube invertedly placed into a swing-out-rotor.

Centrifugation↗

Comparison of the lysis centrifugation and radiometric blood culture systems for recovery of yeast.

The performance of the Isolator (lysis centrifugation) and Bactec (radiometric) detection systems for the recovery of fungi from blood was studied prospectively by comparison of 2,188 paired cultures obtained at two geographically separated teaching hospitals. Eight-three yeast isolates were recovered from 78 (3.6%) cultures that were obtained from 43 patients. Seventy-three (88%) yeast strains were recovered using the Isolator system, and 60 (72%) were recovered in the Bactec system. The average time for recovery of yeast was 2.3 days for the Isolator system and 3.1 days for the Bactec system. Optimal recovery can be accomplished through the use of both systems.

Blood↗

Centrifugal elutriation studies in mast cells from rats, guinea pigs and man.

Elutriation is a cell separation method based on countercurrent centrifugation. For mast cell purification it is superior to density gradient methods since no foreign protein or dense material has to be removed from the purified cells. The elutriation profiles of peritoneal rat mast cells differ considerably if the cells come from non-sensitized or actively sensitized donor animals and/or from different strains. This result is another indication of an altered morphological state of mast cells due to active immunization and is dependent on genetic factors. Cell aggregation proved to be the main obstacle to elutriation purification of guinea pig tissue mast cells. Human mast cells from adenoid tissues, however, could be brought to 70-90% purity, even if the elutriation profiles varied from donor to donor.

Adenoids↗

Density gradient centrifugation of tumor cells from needle biopsies and their respective source tumors: a comparison of density distributions.

A needle biopsy technique was applied to a murine fibrosarcoma (FSa) tumor system. FSa tumors of 8 mm diameter in size were biopsied and then made into single cell suspensions. Resulting density profiles of cells from both sources were compared following centrifugation in Renografin gradients. In all cases, there was excellent agreement between the density profiles of the material from each of the biopsies and the corresponding solid tumors.

Animals↗