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At least 1,441 records · Page 80Linked to original sources
PubMed1978

Separation of multisized drug suspensions into narrow distributions by centrifugal elutriation.

A centrifugal elutriator rotor was used to separate suspensions of micronized hydrocortisone acetate and prednisolone acetate. Up to five distinct particle fractions could beisolated from a single parent suspension, and reasonable reproducibility was exhibited between various elutriator runs. Arithmetic means and associated standard deivations, based on volume, were calculated for each isolated fraction. These data demonstrate the narrowness and uniqueness of each fraction. The particle-size data show an apparent log-normal distribution for each isolate. The uniqueness of each fraction also was demonstrated by dissolution experiments where Fraction 1, the smallest fraction of prednisolone acetate, dissolved much more rapidly than Fraction 4, the largest fraction. The substantial difference in dissolution behavior of these two fractions of prednisolone acetate powder was biopharmaceutically significant.

Centrifugation↗
PubMed1978

Cation-dependent phosphatase activites in a rat pancreatic islet plasma membrane fraction prepared by one-step gradient centrifugation.

A plasma membrane-enriched fraction was prepared from homogenized rat pancreatic islets by a one-step sucrose gradient centrifugation. Using 125I-wheat germ agglutinin as a plasma membrane probe, a fraction was obtained at a sucrose density of about 1.10 that was enriched in 5'-nucleotidase, Mg2+-ATPase and alkaline phosphatase. The fraction contained little, if any, monoamino oxidase activity, insulin or DNA. Hydrolysis of 3-0-methyl-fluoresceinphosphate was stimulated by K+ (10mM) at a pH optimum of pH 8.2. Hydrolysis of ATP-gamma-32P in the presence of MgCl2 was of high specific activity and was optimum at pH 7.0 and 8.2. K+ did not affect ATP-hydrolysis. At pH 8.2, a small fraction of the total Mg2+-ATPase activity was inhibited by ouabain in the presence of Na+ and K+. Since K+-stimulated phosphatase activity does not correlate with Mg2+-ATPase, the two assay systems define separate enzymatic processes.

Adenosine Triphosphatases↗
PubMed1988

Egg-penetration ability and structural properties of human sperm prepared by Percoll-gradient centrifugation.

Human sperm with a high zona-free hamster egg-penetration ability were obtained by centrifuging freshly ejaculated sperm through a discontinuous two-step (47% and 90%) Percoll gradient at 600g at room temperature for 30 min. Highly motile sperm with good penetration ability were recovered in the pellet fraction (Per-sperm), whereas those with low penetration ability were in the gradient interface. The increased penetration ability of Per-sperm, as compared to sperm capacitated by other methods such as a single-tube swim-up or multiple-tube swim-up preparation, was not due to an increased proportion of acrosome reacted sperm. Rather, transmission electron microscopy indicated that Per-sperm were devoid of coating envelopes, which were present around both the head and tail regions of noncapacitated and single-tube swim-up sperm. Changes to the surface of Per-sperm were demonstrated by their decreased interaction with UEA I lectin, which binds specifically to fucose residues. Removal of the coating envelopes as well as other changes on the sperm surface may lead to an enhanced binding of Per-sperm to the oocyte. In addition, 99% of Per-sperm contained chromatin that was fully condensed. By contrast, about 15% of swim-up sperm still possessed incompletely condensed chromatin. With a higher penetration ability, "clean" appearance, and homogeneity of condensed chromatin, Per-sperm are recommended for insemination and studies of human sperm capacitation.

Centrifugation, Density Gradient↗
PubMed1990

Specific binding of 125I-rErythropoietin to Friend polycythemia virus-transformed erythroleukemia cells purified by centrifugal elutriation.

We have used countercurrent centrifugal elutriation (CCE) to determine the distribution of cells with respect to cell volume and buoyant density for an erythroleukemia cell line (JG6) transformed by the polycythemia strain of Friend virus (FV-P), and to determine the effect of inducing the cells to differentiate with dimethylsulfoxide (DMSO) on this distribution. CCE made it possible to obtain suspensions of modal JG6 populations virtually free of dead cells and uniform with respect to volume and buoyant density. These modal populations were assayed for specific binding of erythropoietin (Epo). Between 500 and 550 Epo receptors per cell were detected. These belonged to a single class having a dissociation constant of 0.36 nM. DMSO induction of differentiation of the JG6 cells had no effect on the number of Epo receptors expressed.

Cell Differentiation↗
PubMed1995

Production of senescent cells of Saccharomyces cerevisiae by centrifugal elutriation.

The centrifugal elutriator has been used as a baby machine by loading the chamber with a population of mixed-generation daughter cells and allowing this population to grow, divide and age under continuous washing-out of newborn daughter cells. Clear peaks in the number of elutriated cells were reproducibly obtained for at least ten generations. The parent cells growing in the chamber continued to divide at the steady-state generation time of 95-100 min, showing no change in cycle time during aging. The washed-out daughter cells increased in volume during the first five generations from their steady-state value of 17 micro3 to a maximum of 34 micro3. As to be expected, the generation times of these large daughters, determined in a synchronous batch culture, were shorter (130 min) than that of the steady-state daughters (240 min), even when derived from 15-generation parents. No indication for a volume increase of daughter cells without bud was observed when a population was allowed to grow in the chamber without washing-out the smaller daughter cells. The 15-generation parent population, recovered from the chamber, had an average volume of 80 micro3 and consisted of: (i) 71% cells with more than ten scars, (ii) 13% cells with one to nine scars, and (iii) 17% daughter cells. The production of senescent cells by undisturbed growth in the elutriator chamber has been prolonged to 29 generations. The method is therefore suitable to examine what factors determine the life span of budding yeast.

Cell Cycle↗
PubMed1998

Purification of fibrillin-containing microfibrils and collagen VI microfibrils by density gradient centrifugation.

A method is described for the purification of collagen VI microfibrils and fibrillin-containing microfibrils, respectively. High M(r) microfibril-rich preparations isolated from nuchal ligament by bacterial collagenase digestion and size fractionation were purified by CsCl density gradient centrifugation. Localization of collagen VI and fibrillin within the gradient was achieved by SDS-PAGE/Western blotting. Large collagen VI microfibrillar aggregates were present at the top of the gradient. Hyaluronidase pretreatment dissociated these aggregates and enabled purification of collagen VI microfibrils at a density of 1.33 g/ml. Fibrillin-containing microfibrils separated at 1.37 g/ml and copurified with MAGP1, but not LTBP1, LTBP2, or fibronectin. Confirmation of the intact status of the purified microfibrils was obtained by rotary shadowing. The ability to separate and purify these complex macromolecules provides a powerful means of addressing their molecular composition, organization, and structure:function relationships.

Actin Cytoskeleton↗
PubMed1995

Macrophage activation in response to S-form lipopolysaccharides (LPS) separated by centrifugal partition chromatography from wild-type LPS: effects of the O-polysaccharide portion of LPS.

The S-form lipopolysaccharide (LPS) was effectively separated from a native preparation of smooth-type Salmonella abortus equi LPS by means of the centrifugal partition chromatography (CPC). To clarify the mechanisms by which LPS activates macrophages, CPC-separated S-form LPS was assessed for its ability to induce the secretion of tumor necrosis factor-alpha (TNF-alpha) by murine macrophage-like J774.1 cells in comparison with other fractions of LPS which lacks most of O-polysaccharides. LPS dose-response and time-kinetics studies showed that serum factor(s) regulated especially the onset of TNF-alpha secretion in stimulation with S-form LPS. These results strongly suggest that the native (unfractionated) LPS activates macrophages in both O-polysaccharide/serum-dependent and -independent pathways.

Animals↗
PubMed2001

A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation.

At the end of their growth in the sand fly, Leishmania parasites differentiate into the infective metacyclic promastigote stage, which is transmitted to the mammalian host. Thus, in experimental studies of parasite infectivity toward animals or macrophages, the use of purified metacyclics is generally preferred. While metacyclics of several Leishmania species can be efficiently purified with the aid of lectins or monoclonal antibodies, which differentially exploit stage-specific differences in the structure of the abundant surface glycolipid lipophosphoglycan (LPG), such reagents are unavailable for most species and they are unsuitable for studies involving LPG-deficient mutants. Here we describe a simple density gradient centrifugation method, which allows the rapid purification of infective metacyclic parasites from both wild-type and LPG-deficient Leishmania major. The purified metacyclic promastigotes are authentic, as judged by criteria such as their morphology, expression of the metacyclic-specific gene SHERP, and ability to invade and replicate within macrophages in vitro. Preliminary studies suggest that this method is applicable to other Leishmania species including L. donovani.

Agglutination Tests↗
PubMed1994

Separation of ovine chromaffin granules from lysosomes by successive isoosmolar and hyperosmolar density gradient centrifugation.

Preparations containing ovine chromaffin granules and lysosomes were obtained by differential centrifugation and applied to density gradients. In isoosmolar linear Metrizamide gradients the granules had a lower density than the major portion of the lysosomes (1.05 compared to 1.15); however, in hyperosmolar linear Metrizamide gradients the granule density increased and they migrated close to the lysosomes. The granules separated into two bands on a discontinuous isoosmolar Metrizamide gradient; however, these two bands were similar in terms of granule and lysosomal markers. On a discontinuous hyperosmolar sucrose gradient the granules were more dense than the lysosomes, the reverse of the situation on the Metrizamide gradient. Separation on a discontinuous isoosmolar Metrizamide gradient followed by a 1.8 M sucrose cushion provided a 54-fold purification of granules from lysosomes and similar separations from other subcellular markers. This procedure also provided a 37-fold purification of bovine granules from lysosomes. It was demonstrated that thimet oligopeptidase (EC 3.4.24.15) occurred in the adrenal medulla but is not principally located in the chromaffin granule.

Adrenal Medulla↗
PubMed2002

Highly efficient retroviral gene transfer based on centrifugation-mediated vector preloading of tissue culture vessels.

Efficient retroviral gene transfer into primary cells is a prerequisite for various gene therapeutic strategies. We have developed a transduction protocol based on the preloading of tissue culture vessels with retroviral particles by low-speed (1000g) centrifugation. We show that vector-preloaded tissue culture vessels allow highly efficient gene transfer into various target cells. We obtained transduction rates of up to 85% for primary T lymphocytes after just a single round of transduction. Under clinically relevant conditions using a vector developed for suicide gene therapy and produced under good manufacturing practice (GMP) conditions, the described method allowed generation of large numbers (>2x10(9)) of gene-modified T cells. The preloading concept ensures transduction of target cells in their optimal growth medium regardless of the medium used for vector production. This facilitated highly efficient gene transfer into quite different target cells such as CD34(+) and AC133(+) bone marrow progenitor as well as mesenchymal stem cells. The presented method combines high gene-transfer rates with a great potential for standardization in accordance with GMP guidelines and is consequently well suited for both research and clinical applications. (c)2002 Elsevier Science (USA).

Cell Culture Techniques↗
PubMed1977

The morphology of erythroid cells separated by density gradient centrifugation through Ficoll.

The regenerating blood of geese injected with phenylhydrazine was subjected to large scale, zonal centrifugation through density gradients of Ficoll. In this way, erythroid cells were fractionated according to their respective stages of development. Highly enriched fractions were obtained, containing cells that were well preserved as assessed by both light and electron microscopy. The separated cells exhibited ribosome density and nucleic acid and protein staining patterns typically associated with erythrocyte differentiation. Morphometric analysis of nuclei indicated that despite an apparent net increase in the amount of compact chromatin during development, comparatively little difference existed between the volumes of condensed chromatin present in immature and mature cells. Instead, there was a three fold decrease in nuclear volume between young erythroblasts and reticulocytes, coupled with a concomitant decrease in the volume occupied by dispersed chromatin, RNP and nucleoli. These observations are discussed in relation to molecular changes associated with nuclear differentiation in erythroid cells.

Animals↗
PubMed1978

Protein synthesis by synaptosomes from rat brain: the effect of centrifugal forces.

1. Synaptosomal protein synthesis has been found to differ depending on whether synaptosomes are recovered from the sucrose gradients by centrifuging at 20,000 g or at 150,000 g. 2. The fraction obtained at 20,000 g incorporated 60% more amino acid into proteins after 30 min of incubation than the one obtained in the same period of time at 150,000 g. 3. The respiratory capacity of synaptosomes obtained at 20,000 g in the presence or absence of glucose as substrate, is also better preserved than that of those obtained at 150,000 g. 4. It is shown that the yield, enzymic content, and nucleic acid composition is the same in both fractions. 5. These results cannot be explained by the presence of inhibitory factors in the 150,000 g fraction or by the presence of better preserved free mitochondria in the 20,000 g fraction. 6. Our findings are best explained by postulating that the synaptosomal metabolic activity is damaged when synaptosomes are pelleted at too high a speed.

Acetylcholinesterase↗
PubMed1983

Isolation and purification of plasmids from Bacteroides fragilis using rubidium trichloroacetate density gradient centrifugation.

A rapid and easy final purification method is described for the isolation of plasmids from B. fragilis. Using RbTCA density gradient centrifugation in an airfuge ultracentrifuge ccc plasmid DNA can be separated from RNA, residual chromosomal DNA, linear and oc plasmid DNA. Pure ccc plasmid DNA is obtained from cultures of between 1 ml and 2 l in less than one day.

Bacteroides fragilis↗
PubMed1975

Buoyant density centrifugation of sea urchin embryo chromatin on sucrose-glucose gradient.

Unsheared chromatin isolated from sea urchin embryos was submitted to buoyant density centrifugation in sucrose-glucose gradients. The main peak of blastula chromatin was at a density position of 1.299 plus or minus 0.028 plus or minus 0.009 g ml -minus 1 whereas at gastrula stage a shift to a lower bouoyant density position of (1.276 plus or minus 0.021 plus or minus 0.007 g ml minus 1) was observed. Besides the main peak, a small band with a density of 1.18 g ml minus 1 was noticed. The lighter fraction differed from the heavy one in a higher histone to DNA ratio, a lower proportion of the F-1 histone, and a lower nonhistone to DNA ratio. The most pronounced developmental alterations of proteins were observed at the level of nonhistone protein patterns of the light fractions.

Animals↗
PubMed1981

Studies on osmotic fragility of red blood cells determined with a coil planet centrifuge for workers occupationally exposed to lead.

In order to clarify the relationship between lead exposure level and osmotic fragility of red blood cells determined by the coil planet centrifuge method, several clinical laboratory examinations were performed on 27 male workers employed in a scrap lead refining factory using as controls 40 male workers employed in railway construction. The examinations included measurement of red blood cell and reticulocyte counts, hematocrit, MCV, blood and urine lead concentrations, urine coproporphyrin, and urine delta-aminolevulinic acid. The results were: 1. Osmotic fragility of red blood cells was lower in lead workers at all three hemolytic points compared with the controls. Significant difference was observed in hemolysis of the maximum point (P less than 0.05). 2. The red blood cell and reticulocyte counts, hematocrit value and MCV of the lead workers were not significantly different from those of the controls. Values for blood and urine lead, coproporphyrin, and delta-aminolevulinic acid of the lead workers were much higher than those of the controls (P less than 0.01). 3. In lead workers, close relationships between the osmotic fragility and these laboratory findings were observed: blood lead, r = -0.572, P less than 0.01; coproporphyrin, r = -0.608, P less than 0.01; delta-aminolevulinic acid, r = -0.559, P less than 0.01; urine lead, r = -0.453, P less than 0.05.

Adult↗
PubMed1978

Hemolytic pattern of erythrocytes in the newborn measured by the coil planet centrifuge system and its relationship to neonatal jaundice.

Dynamic osmotic fragility of erythrocytes in cord blood and its changes during the neonatal period were studied by means of a coil planet centrifuge system. The starting-point for hemolysis in the newborn become similar to that of adult blood after approximately a week, while the shift of the end-point to the adult range required one month or more. The percentage of fragile cord blood cells with a hemolysis starting-point above 110 m osmol and the maximum bilirubin level during neonatal period were examined. A high percentage of fragile cells was associated with high bilirubin levels, and when fragile cells comprised more than 7.0% of the total cord blood erythrocytes, the bilirubin level tended to rise above 15 mg/dl.

Bilirubin↗
PubMed1987

A rapid method for the preparation of the neutrophil fraction of granulocytes from human blood by centrifugation on isotonic Nycodenz gradients.

A rapid method for the preparation of neutrophils of the granulocyte fraction of human blood is described. A leucocyte-rich fraction is loaded onto preformed isotonic Nycodenz gradients, formed by mixing a stock isotonic Nycodenz solution with a NaCl diluent solution. After low speed centrifugation a neutrophil fraction of high viability, yield and purity can be isolated from the gradients.

Cell Separation↗