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PubMed1989

Effects of centrifugation on the degradation of short-lived proteins in exponentially growing cultured cells.

The degradation mechanisms of short-lived proteins in cultured cells are unknown, probably due to the lack of procedures which specifically affect the degradation of these proteins. We found that centrifugation of cultured cells, growing either in monolayer or in suspension, between 5000 and 25,000g for 30 min, inhibits (more than 50%) the degradation of short-lived proteins but not of long-lived proteins. Protein synthesis or cell viability is not affected. Centrifugation also disorganizes the Golgi apparatus, as checked by routine electron microscopy, and inhibits the degradation of endocytosed proteins (a lysosomal process which is controlled by the Golgi apparatus). Using different centrifugation speeds, a good correlation was found between alteration of the Golgi apparatus and inhibition of protein degradation.

Animals↗

PubMed1988

A novel system of LDL apheresis combining a centrifugal plasma separator with a specific LDL adsorption column.

A novel system of low density lipoprotein (LDL) apheresis for familial hypercholesterolemia (FH) was developed, combining a centrifugal plasma separator (IBM-2997) with a new adsorption column specific to lipoproteins containing apolipoprotein B (apo-B), and its operation was compared to previous methods. The present system selectively removed LDL without the substantial reduction of high density lipoprotein that was seen with other methods using membrane filters. The capacity for LDL removal was slightly more reduced with the adsorption column than with the membrane filters when a single column was used. Since the ability to obtain plasma in the centrifugal system was much higher than in the system using membrane filters for plasma separation, efficient apheresis could be performed in a much shorter time, without making an arterio-venous shunt. Remixing of blood cells, such as platelets, with the separated plasma sometimes raises the pressure within the plasma component separator, but the problem could be avoided by withdrawing the buffy coat fraction, using the WBC pump of the IBM separator. In conclusion, the novel system, combining the centrifugal plasma separator with the adsorption column, has proved, in our hands, to be a more useful and more convenient method than those previously used for the treatment of severe FH.

Adsorption↗

PubMed1981

Monocyte purification with counterflow centrifugation monitored by continuous flow cytometry.

Continuous monitoring of cell light scatter during counterflow centrifugation of a mononuclear cell suspension allows counting and size recognition of the cell types elutriated. With this method an optimal separation point between monocytes and lymphocytes, determined for each individual donor, may be established. With a constant flow of 15 ml/min this separation point is found at centrifugal velocities ranging from 2348 to 2444 rpm (n = 10). From 50 ml venous blood, 84.1% +/- 4.1% (15.7 +/- 8.6 x 10(6)) of all elutriated monocytes, with a purity of 92.4% +/- 1.4%, is collected in a volume of 50 +/- 1 ml. In the same run, 92% +/- 4.3% of the lymphocytes is gathered in one fraction with a purity of 98.9% +/- 0.7%. After counterflow centrifugation, 91.6 +/- 10.5% of the cells loaded is recovered; viability exceeds 98%.

Cell Separation↗

PubMed1992

Detection of hypodense eosinophils by Percoll multilayer density gradient centrifugation in subjects with normal or slightly elevated eosinophilia. Poor reproducibility and eosinophils of density < 1.077 g/ml.

In patients with marked hypereosinophilia 'hypodense' and 'normodense' eosinophils have been found after density gradient centrifugation. Subsequently this terminology has also been used in studies of patients with milder eosinophilia. However, in these cases the differentiation between normo- and hypodense eosinophils was less clear. This might be due to the high imprecision of the test of density gradient centrifugation, as demonstrated in the first part of this study: the mean within-assay variance of the number of eosinophils in the different density layers was 35%. It was calculated that the test must be performed eight times to obtain an estimate of the true mean for the individual patient. In the second part of the study, the absolute number of 'hypodense eosinophils' in groups of patients with asthma (adults and children) and rheumatoid arthritis (adults) were compared to normal controls. Although a difference in the absolute number of hypodense eosinophils between groups of patients and controls could be demonstrated, the high imprecision of the test of density gradient centrifugation suggested that the technique used was not useful in an individual with normal or slightly elevated eosinophils in the peripheral blood.

Adult↗

PubMed1982

A silicone centrifugation technique for the detection of low parasitaemias of salivarian trypanosomes.

A method using silicone fluid of specific gravity 1.075 was employed to detect low numbers of salivarian trypanosomes in rats infected with T. brucei, T. gambiense, T. congolense or mouse-adapted T. vivax. This method compared favourably with other microsensitive techniques such as the miniature anion-exchange centrifugation and microhaematocrit buffy-coat microscopy methods. The silicone centrifugation technique is based on the density differences between the host's erythrocytes and the parasites. Under the conditions used, the red cells are pelleted by centrifugation through a layer of silicone fluid whereas the trypanosomes remain in the plasma supernatant.

Animals↗

PubMed1986

The combined procedures of Ficoll-Paque centrifugation and anion exchange separation for the recovery of trypanosomes from blood and other tissues.

The conventional method of separating African trypanosomes from infected blood using a DEAE-cellulose (Whatman Chromedia, DE-52) column was modified by the inclusion of an initial centrifugation step employing the density gradient medium, Ficoll-Paque. On centrifugation in this medium using diluted infected rodent blood the trypanosomes moved into the buffy coat interphase which consisted mainly of lymphocytes with a few contaminating RBCs and neutrophils. The buffy coat interphase layer was withdrawn, washed, resuspended and the trypanosomes finally separated from the contaminating blood cells by passage through buffer-equilibrated DE-52 packed in a 25 ml syringe barrel, representing a considerable saving of DE-52 over the conventional method. The centrifugation step was applied to the investigation of other tissues of infected rodents, such as bone marrow, brain, spleen, liver, lymph nodes, kidney and testis, for the presence of trypanosomes. Using the combined procedure, high recoveries of purified brucei group trypanosomes were obtained from bone marrow, brain, spleen and lymph nodes.

Animals↗

PubMed1992

Artificial gravity in Space: vestibular tolerance assessed by human centrifuge spinning on Earth.

Artificial gravity created by the astronauts themselves, without any external power supply, by pedalling on coupled counterrotating bicycles along the inner wall of the space module (Twin Bikes System, TBS), was previously suggested (Antonutto et al., 1991) to prevent musculo-skeletal decay and cardiovascular deconditioning during long term space flights. To investigate whether this unusual rotating environment would determine abnormal stimulations of the vestibular system due to Coriolis cross coupled accelerations, thus leading to acute motion sickness (AMS), the conditions of a rotating environment were reproduced in a human centrifuge. A cycloergometer was fixed to the arm of the centrifuge, the rotation speed of which was equal to that yielding 1 g at the feet level in the TBS (i.e. ranging from 19 to 21 RPM). The ergometer position was such that the combination of the horizontal and gravitational acceleration vectors was 1.414 at the inner ear level and was aligned along the head to feet axis. Three subjects, pedalling at 50 W on a cycloergometer during centrifuge's spinning, were asked to move the head following an AMS' provocation protocol. None of them developed any AMS symptoms. This supports the look of the TBS as tool for avoiding musculo-skeletal and cardiovascular deconditioning during long term space flights.

Adult↗

PubMedDate not supplied

Comparison of gradual and rapid onset runs in a short-arm centrifugation.

A gradual onset run (GOR) in a short-arm centrifugation was performed on ten healthy students. The centrifuge had a 1.8 m radius, and the subjects sat on a chair in a cabin. The Gz force increased to 2.2 Gz at 0.1 degree/sec2 for 32 min. and the same Gz-level was maintained for 20 min. Three out of ten subjects completed the whole protocol; the load on the others was terminated because of symptoms or increased heart rate. There were few symptoms such as vertigo, that was a common problem with a rapid onset run (ROR) in former experiments, due to the short-arm centrifugation. The changes of the flicker test after the load were much less in the GOR protocol than in the ROR protocol, even in the terminated group. GOR seemed preferable to ROR in preventing vertigo even though it took longer to reach the necessary G load.

Acceleration↗

PubMed1985

A comparison between centrifugation and filtration as a means to separate bound and unbound ligand during 3[H]-spiroperidol binding.

A centrifugation method for the separation of bound and unbound ligand during 3[H]-spiroperidol binding in rat brain was compared with the more widely used filtration method. The centrifugation method yielded significantly greater total and specific binding, and lower nonspecific binding, than filtration. Assay reproducibility was also found to be superior when the centrifugation method was used as compared to the filtration binding method. Scatchard plots produced by both methods yielded comparable Kd and Bmax values that were consistent with those previously reported.

Animals↗

PubMed1987

Rapid detection of cytomegalovirus by tissue culture, centrifugation, and immunofluorescence with a monoclonal antibody to an early nuclear antigen.

A rapid, sensitive and specific assay for the detection of cytomegalovirus (CMV) was developed utilizing MRC-5 cells in 24-well plates containing round coverslips. Centrifugation expedited the detection of CMV early antigen with monoclonal antibody. Immunofluorescent staining 16 h after inoculation with a stock CMV preparation (AD-169), demonstrated an 11-fold increase in the number of nuclear inclusions when the specimens were centrifuged (18 +/- 2.2) as compared to the non-centrifuged specimen (1.6 +/- 0.9). However, the number of nuclear inclusions depended on the age of the MRC-5 cells. They were more sensitive to CMV infection between 4 and 11 days after the cells were seeded into plates. Among 159 patient samples cultured for CMV, 23 (14%) were positive by the rapid method (mean of 32 h) and 18 (11%) by routine tissue culture (mean of 12 days). Cytomegalovirus in urine was detected within 1.3 days, whereas buffy coats (2.3 days) and bronchial washings (2.5 days) took longer. Staining for CMV inclusions at more than one time point was necessary for the optimal detection of CMV by the rapid method. We recommend using this assay system as it is rapid, specific, sensitive and versatile for the detection of CMV in many biological specimens.

Antibodies, Monoclonal↗

PubMed1988

Combined use of sonication and monoclonal antibodies for the detection of early and late cytomegalovirus antigens in centrifugation cultures.

A total of 157 clinical specimens was inoculated into shell vials and conventional tube cell cultures containing confluent monolayers of human embryonic lung fibroblasts (HELF). Of 31 clinical cytomegalovirus (CMV) isolates, 30 specimens (96.8%) were positive by the immunofluorescence method on centrifugation vial cultures (CVC-IF), whereas the cytopathic effects (CPE) of CMV were detected in only 14 specimens (45.2%) in conventional tube cell cultures (CCC), P less than 0.001 and in 22 specimens (70.9%) in centrifugation vial cultures (CVC-P), P less than 0.1. Significantly more fluorescent foci were detected in centrifugation cultures inoculated with sonicated urine samples (P less than 0.001). CVC-P is more sensitive than CCC for the diagnosis of CMV (P less than 0.05), and a highly significant difference was observed when we compared the mean day to initial detection of CPE (P less than 0.001). For optimal detection of CMV, both CVC-IF and CVC-P should be used for the laboratory diagnosis of this virus infection.

Antibodies, Monoclonal↗

PubMed1988

Murine cytomegalovirus binds reversibly to mouse embryo fibroblasts: implications for quantitation and explanation of centrifugal enhancement.

In a study of the infection of mouse embryo fibroblasts with murine cytomegalovirus (MCMV), we found that plaque number is directly related to virus concentration and not to the total amount of virus contained in the inoculum. These results suggested that virus binding was reversible and that during infection a binding equilibrium is established which limits the amount of bound virus. Further analysis revealed three categories of plaque based on reversibility after virus adsorption. One group was removed simply by washing cell monolayers after virus removal. A second group of plaques was lost gradually with time, giving complete reversal after 5 min at 37 degrees C. The rate of reversal was temperature dependent, and probably represented true virus dissociation. The final group was irreversible plaques, the number of which increased with increasing infection time. The number of reversible plaques remained constant with time of infection, and represented about 70% of the total plaques after 1 h of virus adsorption. Centrifugation of the virus inoculum onto the fibroblast monolayer at 1000 X g increased plaque numbers up to 100-fold, but had little effect on plaque number when carried out after the virus inoculum was removed. In contrast centrifugation increased the number of reversible plaques, suggesting an increase in the number of virus particles attached to the cell monolayers. We suggest that centrifugation enhances MCMV infection by three mechanisms related to reversibility of binding: (1), it increases the rate of virus association; (2), it decreases the rate of dissociation; (3), by increasing the length of time each virus particle is bound it increases the probability of virus being taken into the cell.

Adsorption↗

PubMed1988

Partial purification of rat kidney iodothyronine-5'-deiodinase by zonal centrifugation.

A rapid and sensitive assay of iodothyronine-5'-monodeiodinase (5'-D) was developed using Sephadex column chromatography for separation of substrate 125I-rT3 from the product free 125I-. The distribution of 5'-D activity on rat kidney cortex cell membranes was examined in isopycnic zonal centrifugation experiments using Na,K-ATPase and NADH-cytochrome C reductase as markers for basolateral and intracellular membranes. 5'-D was mainly distributed on fractions containing endoplasmatic reticulum although some association with basolateral membranes could not be excluded. The isopycnic zonal centrifugation of a microsomal fraction prepared by differential centrifugation purified the 5'-D 8-9 times, 80-90% of membrane-bound 5'-D could be solubilized in fully active form with the detergents CHAPS and C12E8. Solubilization led to a further 2- to 3-fold purification of the enzyme. The soluble preparation was used to characterize 5'-D and as antigens in preparation of monoclonal antibodies for further purification and characterization of 5'-D.

Animals↗

PubMed1995

Enrichment of Penicillium chrysogenum microbodies by isopycnic centrifugation in nycodenz as visualized with immuno-electron microscopy.

A procedure to enrich microbodies from Penicillium chrysogenum and a method to evaluate the purity and integrity of the microbodies are described. As a P. chrysogenum microbody marker acyltransferase (AT) was used. The P. chrysogenum hyphae were converted into protoplasts with Novozym 234. In Percoll-sucrose buffer the protoplasts were separated from mycelial debris after 10,000 x g centrifugation. Purified protoplasts were lysed, and the cell homogenate was centrifuged to form a 14,000 x g pellet. After 2 h, 45,000 x g isopycnic centrifugation of the 14,000 x g pellet on a continuous 20-60% nycodenz gradient, ten fractions were collected. The fractions were analyzed for AT containing microbodies by immuno-blotting and immuno-electron microscopy. The results showed that AT-microbodies are enriched in the 38% nycodenz fraction. The microbodies had a diameter of 400 to 500 nm, revealed an intact single membrane and confined AT. The estimated equilibrium density of the P. chyrsogenum microbodies was 1.20 g ml-1 as deduced from the 38% (w/v) nycodenz concentration.

Cell Fractionation↗

PubMed1984

Characterization of the biophysical properties of human tumor and bone marrow cells as a preliminary step to the use of centrifugal elutriation in autologous bone marrow transplantation.

The principle of centrifugal elutriation (CE) depends on a balance of an outwardly directed centrifugal force and inwardly directed fluid flow and buoyant forces. This method (CE) can be used effectively to separate cells on the basis of size. In the murine model, neoplastic cells from different tumors are generally larger than bone marrow cells and can be removed from bone marrow almost completely with centrifugal elutriation. In order to determine if CE is capable of eliminating human tumor cells from harvested bone marrow (BM), the biophysical characteristics of a variety of human tumor cells and bone marrow cells were determined. Human tumor cells were dispersed into single cell suspensions by several enzymatic digestion and mechanical dissociation methods. The size and density characteristics of these cells were determined with an electronic particle counter and channelyzer and density gradients. Of 40 solid tumors studied, 29 tumors had cell size distributions distinctively larger than BM, as was found in the experimental animal model. The cell size distributions of tumor cells from 11 solid tumors and 7 leukemias were not substantially different from that of BM. Mixtures of BM and cultured human hypernephroma, ovarian, and neuroblastoma cells, were separated into BM and tumor fractions by CE. The separation results as indicated by the labeling index and colony forming efficiency of tumor cells in each fraction showed that a BM fraction virtually free of tumor cells could be obtained. Thus, CE should be able to separate BM cells from most tumor cells metastatic to BM.

Bone Marrow↗

PubMed1987

Rapid detection of cytomegalovirus infections by a tissue culture-centrifugation-monoclonal antibody-biotin/avidin immunofluorescence technique.

A technique using centrifugation, early antigen detection by biotin/avidin fluorescence assay (CEADBAFA) for detection of CMV was compared to a similar method without biotin/avidin (CEADIFA) and to tissue culture alone (TC). The technique using CEADBAFA was the most sensitive of the three assays. The number of CMV-specific fluorescent nuclei was 2-15 fold higher after centrifugation of cell culture monolayers than without centrifugation and the maximum number of positive nuclei was observed after 18 h postinoculation. E-13 was found to be the best of three commercial monoclonal antibodies for identifying CMV positive nuclei. A total of 260 urine, respiratory and genital specimens were tested by both CEADBAFA and TC. Of 46 clinical specimens positive for CMV, 70% were positive by both assays, 7% were positive by TC only and 24% were positive by CEADBAFA only. Two of three specimens positive by TC only could not be evaluated by CEADBAFA because the specimens were toxic to the monolayers on the cover slips. Nearly all of the CEADBAFA positive but TC negative specimens were from patients with a history of CMV infection demonstrated either by TC or serology in other specimens. The CEADBAFA technique should be used for the routine diagnosis of CMV infection.

Antibodies, Monoclonal↗

PubMed2004

Centrifugation-based isolation of myosin for measurement of its synthesis rate in small muscle samples.

Myosin is involved in muscle mobility which is particularly affected in many pathophysiological situations. It is composed of heavy (MHC) and light (MLC) chains and measurements of its specific fractional synthesis rate (FSR) are scarce, mostly because of difficulties in isolating this protein. Our aim was to isolate pure myosin from small rat gastrocnemius skeletal muscle samples by setting up a procedure compatible with determination of stable isotope incorporation into myosin using mass spectrometry detection, allowing calculation of its FSR. A centrifugation method was compared to a validated but time-consuming elution gel electrophoresis method. Statistical analysis by the Bland and Altman test revealed a tight relationship between both methods (r2 >0.97, p <0.0001). The purity of the myosin fractions using the two procedures was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, the centrifugation procedure allowed simultaneous purification of MLC and MHC, whereas the elution gel electrophoresis technique resulted only in MHC isolation. Finally, the FSRs of myosin and MHC were found to be 0.114+/-0.026 and 0.140+/-0.029%/h, respectively (p not significant). In conclusion, the centrifugation method is a useful and reproducible procedure that results in sufficient amounts of pure myosin for reliable determinations of its own synthesis rate in vivo.

Animals↗

PubMedDate not supplied

Effectiveness of centrifuge-induced artificial gravity with ergometric exercise as a countermeasure during simulated microgravity exposure in humans.

To test the effectiveness of centrifuge-induced artificial gravity with ergometric exercise, 12 healthy young men (20.7 +/- 1.9 yr) were exposed to simulated microgravity for 14 days of -6 degrees head-down bedrest. Half the subjects were randomly selected and loaded 1.2 G artificial gravity with 60 W (four out of six subjects) or 40 W (two out of six subjects) of ergometric workload on days 1, 2, 3, 5, 7, 9, 11, 12, 13, 14 (CM group). The rest of the subjects served as the control. Anti-G score, defined as the G-load x running time to the endpoint, was significantly elongated by the load of the centrifuge-ergometer. Plasma volume loss was suppressed (-5.0 +/- 2.4 vs. -16.4 +/- 1.9%), and fluid volume shift was prevented by the countermeasure load. Elevated heart rate and muscle sympathetic nerve activity after bedrest were counteracted, and exaggerated response to head-up tilt was also suppressed. Centrifuge-induced artificial gravity with exercise is effective in preventing cardiovascular deconditioning due to microgravity exposure, however, an effective and appropriate regimen (magnitude of G-load and exercise workload) should be determined in future studies.

Adult↗