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Results for “Centrifugation”

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At least 37 records · Page 2Linked to original sources

PubMed1978

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient.

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.

Acid Phosphatase↗

PubMed1979

Modulation by centrifugation of cell susceptibility to chlamydial infection.

Enhancement of chlamydial infection of cell monolayers by centrifugation was shown to depend on induced cell surface changes. Evidence for this came from analysis of two forms of organism attachment which take place during centrifugation. In 'productive binding', organisms attached to cells and then entered and infected them. In 'unproductive binding', organisms became attached to cells but were not ingested. These organisms could be stripped from the cells by treatment with trypsin and could then infect fresh monolayers. Measurement of attachment kinetics during centrifugation showed that cells passed through three different susceptibility states. Only productive binding occurred in the first 20 min; cells then entered a refractory state during which no attachment took place At about 45 min, attachment recommenced but this allowed only unproductive binding. Induced movement of cell surface structures may enhance infection by promoting specific or non-specific interactions. Failure of ingestion may result from insufficient cell 'receptors' for circumferential binding of the whole chlamydial surface so that engulfment cannot take place.

Cell Membrane↗

PubMed1979

Detection of hepatitis B surface antigen with the miniature centrifugal fast analyzer. A modified reversed passive hemagglutination procedure.

A modified reversed passive hemagglutination test for the detection of hepatitis B surface antigen HBsAg is described. Sera and reagent cells coated with antibody to HBsAg (anti-HBs) are loaded separately into the rotor of a miniature centrifugal fast analyzer. The rotor is centrifuged briefly to transfer the components into its cuvettes. After mixing, the suspensions are allowed to stand at room temperature for 30 min, following which the rotor is again centrifuged and the absorbance of each cuvette is monitored. Cells suspended in serum containing HBsAg leave the light path more rapidly than cells suspended in sera free of antigen. The magnitude of change in absorbance varies directly with the concentration of the antigen. In 45 sera tested by the conventional V-plate technique, findings were as follows: 21 positive, 19 false positive and 5 negative. The automated procedure unequivocally differentiated the 21 positives; results for the false positive and negative specimens were identical and clearly distinguishable from the positive results. The automated procedure enhances specificity, offers equivalent sensitivity, and results that are quantitative and objective.

Absorption↗

PubMed1978

An analysis of the bovine genome by density-gradient centrifugation. Preparation of the dG+dC-rich DNA components.

The dG+dC-rich fractions obtained by density gradient centrifugation of bovine DNA in Cs2SO4/BAMD [J. Cortadas, G. Macaya & G. Bernardi (1977) Eur. J. Biochem. 76, 13--19] were centrifuged in Cs2SO4/Ag+ density gradients. These experiments led to the preparation of the DNA components which had been detected (by analytical centrifugation in CsCl) in the Cs2SO4/BAMD fractions, and also of DNA components which had identical behaviors in Cs2SO4/BAMD gradients and identical buoyant densities in CsCl. A total of eight satellite components and 11 minor components, accounting for 23% and 4% of the bovine genome, respectively, were thus isolated and charcterized in their relative amounts and buoyant densities. The implications of these results on the interpretation of renaturation kinetic data on the bovine genome are discussed.

Animals↗

PubMed1978

New centrifugation blood culture device.

A single-tube blood culture device designed for centrifugation in a tabletop centrifuge is described. Reconstruction experiments using 21 different organisms and human donor blood indicate that excellent recovery can be obtained by centrifugation for 30 min at 3,000 X g.

Bacteria↗

PubMedDate not supplied

A comparative cytologic study of 100 urine specimens processed by the slide centrifuge and membrane filter techniques.

An evaluation of cytology preparations made by the slide centrifuge and membrane filter techniques was made on 100 consecutive urine specimens from 46 patients. The membrane filter technique gave better cellular recovery and produced slides qualitatively superior to those prepared by the slide centrifuge technique. The former, therefore, is the recommended method. The slide centrifuge might be of greater value with urine of low cellularity where the membrane filter has provided only a few to practically no cells. The diagnostic accuracy on clinical follow-up of the patients included in this study is 94% for both methods, with no false positive cases and a 6% false negative rate. This high degree of accuracy indicates that these concentrating methods may lead to clinically valuable information if used routinely in the cytology laboratory.

Adult↗

PubMedDate not supplied

[Homogenization of samples for the culture of mycobacteria. A comparative study of samples with and without centrifugation].

A homogenization method for sputum samples for the culture of acid-fast bacilli that omits the use of centrifugation, passage test tubes and balances is described. 10% sodium phosphate was used as decontaminating substance. This method facilitates the laboratory work since the samples so treated are incubated at 37 degrees C up to the next day when they are then cultured. 676 samples prepared according to this method were compared with samples prepared according to the standard method; a 6,21% positivity in the former compared to a 5,47% in the latter as well as a lower percentage of contamination were obtained. Contamination occurred in 9,91% of samples without centrifugation and in 14,35% of centrifuged samples.

Bacteriological Techniques↗

PubMed1979

The calculation of some physical parameters of proteins from sucrose density gradient centrifugation data.

A simplified procedure is described for the calculation of protein physical parameters from data obtained by sucrose density gradient centrifugation. The position of an uncharacterized protein relative to standard proteins is determined after centrifugation through linear 5-20% sucrose gradients prepared in both H2O and D2O. From these data, together with the density of each 5% sucrose buffer, the partial specific volume and standard sedimentation coefficient can be calculated without knowledge of rotor dimensions, angular velocity, time of centrifugation, or solvent viscosity. In the absence of specific solvent-induced changes in protein composition or conformation, the magnitude of error due to the approximations inherent in the method is estimated to be less than 2.3% in partial specific volume or less than 10-15% in sedimentation coefficient.

Centrifugation, Density Gradient↗

PubMed1979

Density gradient centrifugation for separation of different stages of Toxoplasma gondii.

Suitable procedures for separation of various stages of Toxoplasma gondii by density gradient centrifugation have been delineated using peritoneal exudate of infected mouse as a model. Separation from host cells was effected by gradient centrifugation at 450 g for 30 minutes. Using Ficoll, dextran and sucrose, average parasite recoveries by pooling up to and including the peak fraction of 73.65%, 66.18% and 65.68% respectively were obtained. Toxoplasma trophozoites peaked at density of 1.040 g/ml with Ficoll, 1.060 g/ml with dextran and 1.110 g/ml with sucrose. In view of successful separation of exo-enteric stages of Toxoplasma by density gradient centrifugation, possible application of this method to isolation of various endo-enteric stages is discussed.

Animals↗

PubMed1979

Improved method for detecting hemagglutination by centrifugal analysis.

Centrifugal analysis can be used to detect hepatitis B surface antigen, antibody to rubella virus, and fibrin-related antigen. The procedure is performed with the same reagents used in conventional hemagglutination studies. Positive and negative reactions are distinguished by the rates of erythrocyte clearance in the centrifugal field (delta A/delta time); positive cells move more rapidly than negative cells, and this difference varies directly with the concentration of detectable antigen or antibody. This phenomenon is thought to be a result of the greater adhesion of negative cells to the cuvette's surface. Sensitivity and specificity are greater in the centrifugal analysis technique than in the more conventional hemagglutination tests. False-positive reactions are eliminated and the quantitative data are accurate and reproducible.

Antibodies↗

PubMedDate not supplied

Centrifugal Cytology, IV. The Prearation of fixed stained dispersions of gynecological cells.

The Centrifugal Cytology technique has been utilized to produce glutaraldehyde fixed stained dispersions of both conventional Ayre scrapes and Davis pipet (PAPette) samples. Light microscope studies of dispersions of both types of cells on conventional microscope slides indicated that both the tinctorial and morphological appearance of the cells after Papanicolaou staining was very similar to that observed with conventional smears and that the same criteria could be utilized with the Centrifugal Cytology dispersions to screen the cells for cancer as had previously been used with the smears. A preliminary study indicated that six out of six positives with no false negatives or false positives were found. The Centrifugal Cytology technique appears to have promise as a method for preparing suspension samples such as pipets of gynecologic cells. Scanning electron microscope studies reveal that the squamous epithelial cells are very thin and at least some of them are covered by a network structure.

Carcinoma, Squamous Cell↗

PubMed1979

Improved methodology for the quantitative analysis of mixtures by band centrifugation: reversible equilibration in immunoglobulin preparations.

Double-sector aluminium centerpieces are modified for use in analytical high-speed band centrifugation up to 60,000 rpm. A method has been developed for testing IG preparations according to administrative regulations with special regard to precision and reproducibility. The proposed band-forming centrifugation method with scanner registration in UV absorption optics and special evaluation of the curves gives values in comparison to well-known procedures by Svedberg and Pedersen. Subjective and systematic errors are especially estimated with this method and the sensitivity is best suited to automatic computerization and results are therefore readily available at the end of the centrifuge run within a few minutes. Investigations have been undertaken to analyze reversible equilibration in immunoglobulin preparations; the results are discussed.

Centrifugation, Density Gradient↗

PubMed1978

Lecithin/sphingomyelin ratios centrifuged and uncentrifuged amniotic fluid.

The literature suggests that the presence of cellular debris and sediment in amniotic fluid samples may alter the value of the lecithin/sphingomyelin (L/S) ratios, with a loss of as much as 75 percent of the surface-active lecithin1 and consequently a marked change in the L/S ratio. We wish to report our findings on 22 amniotic fluids obtained by transabdominal amniocentesis and analyzed with and without preliminary centrifugation. We find the L/S ratio is indeed unaltered by centrifugation. However, the actual amounts of lecithin and sphingomyelin appear to be lower in centrifuged samples.

Amniotic Fluid↗

PubMed1979

Relationship between photoreceptor terminations and centrifugal neurons in the optic lobe of octopus.

Retinal bundles, connecting the retina of the octopus to the ipsilateral optic lobe, contain both retinal photoreceptor axons that terminate in the optic lobe and centrifugal axons whose cell bodies lie within the lobe. Staining axonal elements in proximal stubs of individual retinal bundles by cobalt diffusion and subsequent sulphide treatment reveals the topographic relationship between afferent terminals and centrifugal cell bodies. At the outer border of the plexiform layer, stained terminal bags (photoreceptor axon enlargements), an indicator of photoreceptor terminal spread within this layer, overlap stained centrifugal cell bodies located within the inner granule layer. The details of this overlap indicate a dorsoventral representation of each retinal bundle within the optic lobe cortex.

Animals↗

PubMed1979

High concentration active enzyme centrifugation: analysis of active polymeric forms at up to 10 000-fold higher concentrations than with conventional methods.

This paper describes the theoretical basis, experimental technique, and experimental evaluation of a new method of analysis called "high concentration active enzyme centrifugation". It extends by up to four orders of magnitude the upper concentration limits at which the technique of "active enzyme centrifugation" can be used for analysis of enzyme structure. This new theory is largely based on certain properties of Gaussian curves which we have described in previous publications [Wei, G.J., & Deal, W.C., Jr. (1976) Anal. Biochem. 75, 113-121; Anal. Biochem. (1978) 87, 433-446]. One of the most important aspects of this development is that it extends the concentration range upward so that experiments can be performed on enzymes in the active polymeric forms corresponding to their in vivo states. Furthermore, this expansion includes the range in which most enzymes go through all their association-dissociation transitions from one polymeric form to another. Hence, the method can be used to define the various concentration-dependent transitions and also to ascertain which of the various polymeric forms of an enzyme are active, under various conditions. This method also retains the many favorable characteristics inherent in the active enzyme centrifugation technique. In studies with lactate dehydrogenase, the results from this method of band sedimentation were identical within experimental error (about 1.5%) with results from conventional boundary sedimentation velocity studies.

Animals↗

PubMed1975

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↗

PubMed1976

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↗

PubMed1978

Centrifugal method of determining red cell deformability.

A recently developed technique for deforming red blood cells (RBC) in which they are centrifuged through buffer and into a glutaraldehyde solution was evaluated as a method of assessing cellular deformability (i.e., the ability of the entire RBC to form a new configuration). To accomplish this, RBC populations of differing cellular deformability were tested, using three generally accepted techniques to obtain these differences: partial fixation with low concentrations of glutaraldehyde, density fractionation, and suspension of RBC in nonisotonic media. Our results indicate that at a constant deforming force the mean deformed length of the RBC decreased under conditions where cellular deformability is known to decrease, thus suggesting the usefulness of this centrifugal method for the estimation of this cellular property.

Centrifugation↗