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PubMed1991

Large-scale purification of inactivated hepatitis A virus by centrifugation in non-ionic gradients.

Formalin-inactivated hepatitis A virus (HAV) can be purified for vaccine preparation by centrifugation in Renografin-76 (diatrizoate meglumine and diatrizoate sodium) gradients. Both continuous-flow rate-zonal and isopycnic methods were used for the separation of a major antigen component from minor antigen and host protein. The major antigen component, which appeared to contain complete virions by electron microscopy, could be recovered from gradients and accounted for approximately one third of the total antigen in the starting material. The HAV-specific purified antigen could be enriched 200-300-fold by either centrifugation procedure. The purified HAV antigen, when adsorbed to alum and inoculated into mice, was found to be highly immunogenic.

Antigens, Viral↗
PubMed1992

Comparison of centrifugation methods for molecular and morphological analysis of membranes associated with RNA replication of the flavivirus Kunjin.

Kunjin virus-infected cells were lysed and the cytoplasmic extract was subjected to sedimentation analysis. After centrifugation at 16,000 x g for 10 min about 70% of the original RNA-dependent RNA polymerase (RDRP) was recovered in the pellet; most of this enzymic activity was recovered in the soluble fraction after treatment with NP40 detergent. Membrane fractions were prepared from cytoplasmic extracts by centrifugation in discontinuous density gradients comprising w/w or w/v sucrose solutions, either for 3 h (top-loaded on 4 ml 20-60% sucrose) or for 19 h (centre-loaded in 37 ml 0-60% sucrose). Similar separations of bands of light membranes were obtained in all gradients. Multi-layered heavy membrane bands obtained with w/w sucrose gradients were resolved into two well-separated bands (F4 and F5) using w/v sucrose gradients. Thin-section electron microscopy of embedded membrane fractions, gel analysis of intracellular RNA, and RDRP assays showed that the w/w centre loading method and the w/v top-loading (short spin) method produced similar recoveries and distributions of smooth and rough membranes, intact virus particles and RDRP activity. The distribution of intracellular viral RNA and proteins was coincident with the RDRP, all being located in the F4 and F5 bands which contained the characteristic membrane structures induced during flavivirus infection. Significant advantages of the preferred method (w/v sucrose, top loading and short spin) were its rapidity, good preservation of membranes and RDRP, and the concentrations of RDRP achieved in the small volume fractions collected from a total of 4.5 ml.

Cell Membrane↗
PubMed1988

Differential effects of tri-n-butyltin chloride on macromolecular synthesis and ATP levels of rat thymocyte subpopulations obtained by centrifugal elutriation.

Using centrifugal elutration, rat thymocytes were separated into three fractions by size. Fraction 1 (F1) consisted of a large population (greater than 88% of all cells) of small, nonproliferating thymocytes. Fraction 2 (F2; 7% of all cells) was enriched in medium-sized thymocytes and showed an increased macromolecular synthesis. In the small fraction 3 (F3; less than 5% of all cells), large thymocytes, monocytes, granulocytes and cells in mitosis were concentrated. F3 demonstrated the highest proliferative activity. Fractions were characterized by size, cell morphology, DNA, RNA and protein synthesis and steady state ATP levels. The effects of the inhibitor of oxidative phosphorylation, tri-n-butyltin chloride (TBTC) on ATP levels and the incorporation of DNA, RNA and protein precursors were investigated for each thymocyte fraction. Although ATP levels increased with increasing thymocyte volume, TBTC reduced ATP levels in each subfraction proportionally. The incorporation of thymidine and leucine was markedly reduced in all fractions by TBTC, but not to the same extend. Dependent on the TBTC concentration, the rapidly proliferating cells of F2 and F3 were less affected compared to the noncycling cells of F1. The incorporation of uridine in the unfractionated cells and in F2 and F3 was also decreased by TBTC. However, at concentrations between 0.1 and 1 microM, TBTC stimulated uridine incorporation in the small thymocytes of F1. Therefore, fractionation of thymocyte suspensions was necessary to detect differential effects of TBTC on subpopulations. Centrifugal elutriation was found to be a useful tool with which to obtain subfractions of isolated rat thymocytes.

Adenosine Triphosphate↗
PubMed1988

Effect of microtubule reactive drugs on steroid- and centrifugation-induced germinal vesicle migration during goldfish oocyte meiosis.

During the process of progestogen-induced meiotic maturation in the goldfish oocyte, the oocyte nucleus (germinal vesicle, GV) migrates to the sperm entry site or micropyle at the animal pole. Following GV migration (GVM) to the micropyle, the nuclear membrane undergoes dissolution (GVD) and the cell enters metaphase I in preparation to generate the first polar body. Microtubule destabilizing drugs including colcemid, nocodazole and vinblastine were found to elicit GVM, mimicking the process which occurs just prior to the prophase I-metaphase I transition during steroid induced oocyte meiotic maturation. In addition, these drugs enhanced the induction of GVM by 17 alpha, 20 beta dihydroxy-4-pregnen-3-one, a potent, naturally occurring meiotogenic steroid in this species. By contrast, taxol, a microtubule stabilizing drug, was found to inhibit steroid induced GVM. A new assay for centrifugation induced GVM was applied to the goldfish oocyte in order to assess effects of steroids and drugs on GVM, without the complication of GVD or the restrictions imposed by the slow time course of naturally occurring GVM. The effective centrifugal force (ECF) required to elicit GVM in 50% of the oocytes (ECF50) decreased significantly after short incubations (1-5 hr) of oocytes with either 17 alpha,20 beta dihydroxy-4-pregnen-3-one or microtubule disrupting drugs (i.e., colcemid, nocodazole, or vinblastine). A working hypothesis, modeled after the effects of microtubule disrupting agents on intermediate filament arrays in somatic cells, is proposed in which a small number of microtubules or other polymeric tubulin units are responsible for maintaining a cytoskeletal array.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1996

Performance of a centrifugal phytotron.

It is possible to cultivate plants under an artificial gravity field generated by a centrifugal device in space. In order to determine an optimal magnitude of gravity, there is a need to investigate the relationship between plant and growth and gravity, including not only reduced gravity but also gravity greater than 1G. A prototype centrifugal phytotron was designed and fabricated in order to investigate the relationship between plant growth and increased gravity. This device enables us to cultivate plants over the long term by controlling environmental conditions in the phytotron such as temperature, relative humidity, CO2 concentration and light intensity. The results of our experiment indicate that plant seeds can germinate and grow even under an artificial gravity which changes sinusoidally from 2G to 4G.

Acceleration↗
PubMed1976

Dependence of the sedimentation of high molecular weight DNA on centrifuge speed.

A theory by Zimm [B.H.Zimm, Biophys. Chem. 1-(1974)279] predicts that for a given centrifuge speed, there is a broad maximum in a plot of the sedimentation coefficient of DNA against molecular wight. Experimental measurements of these maxima for various centrifuge speeds were made for double-helical DNA in neutral sucrose gradients and singlestrand DNA in alkaline gradients. The measurements are in quantitative agreement with the theory, providing good evidence for its validity. The existence of the maximum shows that there is a limit to the sedimentation rate under specified conditions for KNA in the linear form. By implication, DNA observed to sediment faster than this limit is not in the linear form to which most sedimentation theory is applicable.

Centrifugation, Density Gradient↗
PubMed1987

Binding of renin and angiotensinogen to macromolecules in plasma studied by sedimentation equilibrium centrifugation.

Plasma from normal and aggressive mice was subjected to sedimentation equilibrium centrifugation in an air-driven centrifuge. The aim was to study the apparent molecular weight of renin and angiotensinogen in undiluted plasma. Both renin and angiotensinogen appeared heterogeneous with respect to molecular size, suggesting binding to plasma macromolecules. Subsequent high pressure liquid chromatography, using a size exclusion column, demonstrated molecular homogeneity and molecular weights as found in noncentrifuged plasma, indicating that the binding is easily reversible. It is concluded that renin and angiotensinogen in undiluted and unfractionated plasma are weakly bound to plasma macromolecules. This may reduce their activity and to some extent explain the previously observed apparent inhibition of the enzymatic activity.

Aggression↗
PubMed1990

Evaluation of cell subpopulations isolated from human tumor xenografts by centrifugal elutriation.

Human epidermoid tumor cells (Coll2, ME180, A431, HEp3) grown as xenografts in nude mice, were dissociated into single cell suspensions using an enzyme cocktail containing 0.025% collagenase, 0.05% pronase, and 0.04% DNase. The dissociated cell suspensions were separated by centrifugal elutriation into fractions containing homogeneous cell subpopulations primarily based on the differences in the rates of sedimentation. The quality of separation was evaluated by several techniques including flow cytometry, cell volume distributions, in vitro colony forming assay and morphological examination of Wright-Giemsa stained cells. In each separated fractions, the host to neoplastic cell ratio, the DNA ploidy, the plating efficiency and the cell cycle distribution were determined. After an initial separation of non-neoplastic host cells from malignant cells, a purity of greater than 95% host cells was obtained from the four xenografts studied. DNA analysis of tumor suspensions showed that neoplastic cells of different xenografts contained aneuploid cells with a DNA index of 1.51 to 1.95. The neoplastic cells were further separated into fractions according to their positions in the cell cycle. Fractions containing greater than 95% G1, 65% S, and 72% G2M cells were obtained from HEp3 xenografts. Less efficient separation with respect to cell cycle was attained with cells derived from Coll2, ME180, and A431 xenografts. Colony forming abilities of the neoplastic cells were determined at different phases of the cell cycle and found to be similar to those of the unseparated cell suspensions after corrections for non-neoplastic host cells were made. These investigations indicate that centrifugal elutriation is an effective technique of obtaining homogeneous subpopulations of cells from human tumor xenografts for various tumor biology and cell kinetics studies.

Animals↗
PubMed1987

Comparison of MRC-5 and HFF cells for the identification of cytomegalovirus in centrifugation culture.

Detection of cytomegalovirus (CMV) by centrifugation culture was compared in MRC-5 and HFF cells. At 24 hr 47 of 130 specimens (36%) were positive for CMV, 45 in MRC-5, and 35 in HFF cells. At 48 hr 45 specimens were positive in both MRC-5 and HFF cells. MRC-5 cells appear to be superior for the detection of CMV at 24 hr in centrifugation culture; however, both lines are comparable at 48 hr.

Cell Line↗
PubMedDate not supplied

Evaluation of immunofluorescent reagents, centrifugation, and conventional cultures for the diagnosis of adenovirus infection.

In this study we have evaluated four fluorescent antibody reagents, three monoclonal and one polyclonal, for identification of adenovirus isolates and compared four conventional cultures (human embryonic kidney, A549, HEp-2, and MRC-5 cells) with centrifugation culture for rapid diagnosis. For identification of adenovirus isolates by immunofluorescence, CDC reagent and Adenoclone, both monoclonal antibodies to the hexon group-reactive antigen, were more sensitive and easier to interpret than the other two reagents tested. HEK and A549 cells were the most sensitive for isolation of adenovirus. Although A549 cells were an inexpensive alternative to HEK, A549 cell monolayers deteriorated more rapidly and passages were more often required. Centrifugation cultures with A549 cells detected 77% of positives within 2 days and 100% within 5 days, whereas isolation in conventional culture required up to 10 days for HEK and up to 20 days for MRC-5 cells.

Adenoviridae Infections↗
PubMed1989

Lack of insulin stimulation on Percoll-prepared or high-speed-centrifuged rat liver hepatocytes.

Rat liver hepatocytes isolated from a 30-31% percoll density gradient at 10,000g are refractory toward insulin stimulation of 14CO2 formation and 14C-incorporation into protein from [2,3-14C]succinate. Basal hepatocyte oxidation of succinate was not impaired by the presence of 5% percoll in the incubation medium nor was it impaired when percoll-free hepatocytes were used that had been isolated after centrifugation at 9000g; however, in both instances the stimulatory effect of insulin was lost. Hepatocyte damage may have occurred in these processes. This is in contrast to previous work which shows that insulin (10 mU/ml) will stimulate [2,3-14C]succinate oxidation and [2,3-14C]succinate carbon incorporation into protein in non-percoll-treated hepatocytes (isolated by centrifugation at 10g) by about 29%. We conclude that the latter procedure although more time consuming is the more gentle method of choice and leaves the hepatocyte in a form more closely related to an in vivo state than does treatment with a percoll density gradient at 10,000g.

Animals↗
PubMed1991

Measurement of IgM responses to a subunit influenza A vaccine by sucrose-gradient centrifugation and membrane-filtration enzyme immunoassays.

IgM responses to a deoxycholate-split influenza vaccine containing the surface antigens of the H3N2 virus A/Philippines/2/82 were studied in five volunteers, three of whom were seronegative by haemagglutination inhibition (HI) tests. Responses were measured by a sucrose-gradient centrifugation technique, in which IgM-specific HI activity was computed as a proportion of total IgM and IgG-specific HI activity, and by a membrane filtration-enzyme immunoassay (MF-EIA). Responses could be detected in all volunteers when measured by sucrose-gradient centrifugation within 1-2 weeks, and the IgM induced was 5-40% of total HI-specific activity. The response was also observed in the presence of low levels of pre-existing antibody. Levels of IgM antibody, when measured by MF-EIA, could be easily detected in two of the volunteers, while those of two others were very low and there was no response in a fifth. No biphasic virus-specific response to vaccination, involving first IgM and then IgG, could be measured by either technique. From these studies, the sucrose-gradient fractionation technique appears to be the more sensitive procedure.

Centrifugation, Density Gradient↗
PubMed1992

Optimized centrifugation for rapid elution of DNA from agarose gels.

A simple method has been developed for the isolation of DNA from agarose gels. Centrifugation in a low-cost device for 45 s at low speed provides a high yield of DNA suitable for further manipulation by restriction enzymes, T4 DNA ligase, Taq polymerase, Klenow fragment, and T4 polynucleotide kinase, and also for sequencing. In contrast, centrifugation for greater than 1 min leads to coelution of substances that inhibit DNA ligase.

Blotting, Southern↗
PubMedDate not supplied

Artificial gravity--head movements during short-radius centrifugation: influence of cognitive effects.

Short-radius centrifugation is a potential countermeasure against the effects of prolonged weightlessness. Head movements in a rotating environment, however, induce serious side effects: inappropriate vestibular ocular reflexes (VOR), body-tilt illusions and motion sickness induced by cross-coupled accelerations on a rotating platform. These are well predicted by a semicircular canal model. The present study investigates cognitive effects on the inappropriate VOR and the illusory sensations experienced by subjects rotating on a short-radius centrifuge (SRC). Subjects (N=19) were placed supine on a rotating horizontal bed with their head at the center of rotation. To investigate the extent to which they could control their sensations voluntarily, subjects were asked alternatively to "fight" (i.e. to try to resist and suppress) those sensations, or to "go" with (i.e. try to enhance or, at least, acquiesce in) them. The only significant effect on the VOR of this cognitive intervention was to diminish the time constant characterizing the decay of the nystagmus in subjects who had performed the "go" (rather than the "fight") trials. However, illusory sensations, as measured by reported subjective intensities, were significantly less intense during the "fight" than during the "go" trials. These measurements also verified an asymmetry in illusory sensation known from earlier experiments: the illusory sensations are greater when the head is rotated from right ear down (RED) to nose up (NU) posture than from NU to RED. The subjects habituated, modestly, to the rotation between their first and second sequences of trials, but showed no better (or worse) suppression of illusory sensations thereafter. No significant difference in habituation was observed between the "fight" and "go" trials.

Adaptation, Physiological↗
PubMed2004

Two distinct methods analyzing chromatin structure using centrifugation and antibodies to modified histone H3: both provide similar chromatin states of the Rit1/Bcl11b gene.

Chromatin state of a 2-Mb region harboring Rit1/Bcl11b on mouse chromosome 12 was examined using two distinct methods. One is ChIP assay examining the degree of enrichment with histone H3 methylated at lysine 9 (H3-mLys9) in chromatin and the other is H/E (heterochromatin/euchromatin) assay that measures a chromatin condensation state by using centrifugation. The ChIP assay showed that a 50-kb interval covering the gene and an upstream region constituted chromatin enriched with unmethylated H3-mLys9 in cells expressing Rit1 compared to cells not expressing Rit1. In contrast, regions other than the 50-kb interval did not show much difference in the enrichment between the two different types of cells. On the other hand, H/E assay of two expressing and two non-expressing tissues provided compatible fractionation patterns, suggesting that the chromatin condensation state detected by H/E assay is correlated with the chromatin state controlled by histone H3 tail modification linked to gene expression. These results indicate that the centrifugation-based H/E assay should provide a new approach to the regulation of chromatin structure with respect to its condensation state, complementing ChIP assays.

Animals↗
PubMed2005

Extrusion and removal of lipid from the cytoplasm of porcine oocytes at the germinal vesicle stage: centrifugation under hypertonic conditions influences vitrification.

In the present study, we examined a novel lipid removal method, centrifugation in solutions made hypertonic by adding 0.27 M sugar. This allowed the lipid to be extruded and removed without the loss of active mitochondria or extra cytoplasm. The type of sugar influenced the proportion of oocytes that could be stratified by centrifugation. Glucose induced the highest extrusion rate of lipid droplets. After vitrification the rates of survival, germinal vesicle breakdown and metaphase II were 30, 26, and 7%, respectively, for lipid-removed GV oocytes; this was significantly higher (P<0.05) than for corresponding vitrified lipid-intact oocytes (2, 0, and 0%, respectively). These results indicated that this method is useful to remove whole lipid droplets without losing mitochondria and improves cryotolerance of porcine GV oocytes.

Animals↗
PubMed2006

Physical stability, centrifugation tests, and entrapment efficiency studies of carnauba wax-decyl oleate nanoparticles used for the dispersion of inorganic sunscreens in aqueous media.

Aqueous nanoscale lipid dispersions consisting of carnauba wax-decyl oleate mixtures acting as carriers or accompanying vehicles for inorganic sunscreens such as barium sulfate, strontium carbonate, and titanium dioxide were prepared by high pressure homogenization. For the manufacture of these nanosuspensions, three pigment concentrations (%wt), namely 2, 4, and 6, and two carnauba wax-decyl oleate ratios, 1:1 and 2:1, were used, being some of these combinations chosen for stability studies. Six-month physical stability tests at 4, 20, and 40 degrees C selecting the mean particle size and the polydispersity index of the nanosuspensions as reference parameters were performed. Centrifugation tests of the nanosuspensions assessed by transmission electron microscopy and by the determination of the content of pigments and carnauba wax in the separated fractions were done. The mean particle sizes and the polydispersity indices of the nanosuspensions were not altered after six-month storages at 20 and at 40 degrees C. However, the storage of those at 4 degrees C considerably increased the particle size and polydispersity of the systems, particularly when wax-oil ratios (2:1) were used for the entrapment of the pigments. Transmission electron micrographs of centrifuged samples denoted the presence of three major fractions showing the different types of particles integrated into the nanosuspensions. Furthermore, it was observed that not all the carnauba wax participated in the entrapment of the pigment. Regarding the amount of pigment being encapsulated or bonded by the wax-oil matrices, entrapment efficiencies higher than 85.52% were reported.

Centrifugation↗