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PubMed1990

Settling-time dependence of rat bone marrow cell partition and counter-current distribution in charge-sensitive aqueous two-phase systems. Relationship with the cell partitioning mechanism.

Differences in the settling-time dependence of single and multiple cell partitions have been found between heterogeneous (bone marrow cells) and homogeneous (erythrocytes) populations when using charge-sensitive dextran-poly(ethylene glycol) aqueous two-phase systems. The cell populations were partitioned using both single test-tube experiments and multiple thin-layer counter-current distribution. Lengthening the settling time, to favour phase separation, and decreasing the upper phase volume are more effective in fractionation by the counter-current distribution of heterogeneous cell populations than increasing the interfacial tension, although all three were employed to speed phase settling. On the basis of these results, the original cell partitioning mechanism proposed for non-charge-sensitive systems has been extended to charge-sensitive systems.

Animals↗

PubMed1989

Heterogeneity of a crude synaptosomal preparation, studied by affinity partitioning using hexaethonium-poly(ethylene glycol).

The heterogeneity of a synaptosomal preparation was studied by the use of affinity partitioning in combination with centrifugal counter-current distribution. Hexaethonium-poly(ethyleneglycol) was used as the extracting agent. The fractions were analyzed for: light scattering, protein, choline acetyltransferase, L-glutamate decarboxylase, glutamine synthetase, 2',3'-cyclicnucleotide-3'-phosphohydrolase, acetylcholinesterase and succinate dehydrogenase. The material was fractionated into three main fractions which differed in their content of marker-enzymes.

Animals↗

PubMed1994

Biochemical characterization of human erythrocytes fractionated by counter-current distribution in aqueous polymer two-phase systems.

The fractionation of normal human erythrocytes by counter-current distribution (CCD) in charge-sensitive dextran-polyethylene glycol two-phase systems was confirmed and extended to red blood cells from heterozygous beta-thalassaemic patients. The differences between the distribution profiles of normal (homogeneous) and abnormal (heterogeneous) red blood cells reflect their different surface-charge properties. As suggested by the decline of membrane sialic acid released after neuraminidase treatment and the specific activities of two age-dependent enzymes (membrane acetylcholinesterase and intracellular pyruvate kinase) in the distribution profiles (from the left- to the right-hand side fractions), the fractionation seems to be according to red blood cell age. A constancy of the 2,3-bisphosphoglycerate level was observed in ageing red blood cells.

2,3-Diphosphoglycerate↗

PubMed1978

Portal circulations and their relation to counter-current systems.

We have reviewed the distribution of portal circulations throughout the animal body; they are commoner than is generally supposed. Most portal circulations consist of two serial capillary beds connected by one or more larger vessels. We have called these 'convergent' portal circulations: examples are hepatic portal, placental, hypophysial, renal, ovarian and testicular circulations, as are parts of the lymphatic circulation. A second type of portal circulation, which is less common, consists of two serial capillary beds that are not connected by larger vessels. These we have called 'continuous' portal circulations: adrenal and pancreatic circulations are examples of this type. When a countercurrent concentrating mechanism exists in the body it is always part of the primary or secondary bed of a convergent portal circulation, though some convergent portal circulations are not associated with countercurrent mechanisms.

Adrenal Glands↗

PubMed1981

Separation by counter-current distribution of rat and chicken erythrocytes of different age, and its application to the assay of enzyme activities.

The separation of cells with different ages from erythrocyte populations of adult rats and young or adult chickens have been achieved by counter-current distribution (CCD). A thin-layer CCD apparatus has been employed. Erythrocytes from blood samples taken at different times after 59Fe i.p. injection were separated by CCD. By compilation in a "composite curve" of the hemoglobin and radioactivity CCD profiles obtained for each erythrocyte population, the distribution of cells according to age can be inferred. Young erythrocytes of rats are located at the right part of the CCD curves, while older cells are distributed towards the left. An opposite distribution has been found for erythrocytes from adult or young chickens. As a first attempt for the application of the CCD procedure to the assay of enzyme activities, it was found a decrease in phytase activity as the age of chicken erythrocytes increases and an increase in phosphoglycerate kinase and phosphofructokinase as the age of rat erythrocytes increases.

6-Phytase↗

PubMed1978

Biochemical studies on rat liver Golgi apparatus. III. Subfractionation of fragmented Golgi apparatus by counter-current distribution.

Vesicular fragments of Golgi apparatus, smooth- and rough-surfaced microsomes from rat liver are differently partitioned in aqueous polymer two-phase systems consisting of dextran, polyethylene glycol, and sodium phosphate buffer. At a given polymer concentration, the amount of material partitioned in the top phase increases in the following order: rough microsomes less than smooth microsomes less than Golgi fragments. Counter-current distribution of Golgi fragments in the system consisting of 6.8% (w/w) dextran T500 and 6.8% polyethylene glycol 4,000 results in the separation of the fragments into three fractions; i.e. Fractions I, II, and III. NADH- and NADPH-cytochrome c reductase activities are detected almost exclusively in Fraction I, whereas the activities of galactosyltransferase, acid phosphatase, 5'-nucleotidase, and thiamine pyrophosphatase are maximal in Fraction III and minimal in Fraction I. The distribution of these enzymes suggests that Fraction I is similar to, though not identical with, microsomes, Fraction III resembles plasma membrane and lysosomes, and Fraction II is between the two. It is concluded that NADH- and NADPH-cytochrome c reductases are localized in a restricted region of the Golgi structure and that intra-Golgi differentiation seems to proceed in a discontinuous manner.

Acid Phosphatase↗

PubMed1988

Partitioning behaviour in aqueous two-phase systems and fractionation by counter-current distribution of chick-embryo erythrocytes with numerical resolution of distribution curves.

The partition of chick-embryo and young-chick erythrocytes in dextran-poly-(ethylene glycol) two-phase systems depends on the interfacial tension and electrical potential differences between the phases. Counter-current distribution with charged 5% dextran-poly(ethylene glycol) systems has proved to be an adequate method for the separation of primitive and definitive erythrocytes present in chick embryos when a phase settling time of 20 min is used. The computer-aided numerical resolution of experimental curves has shown the existence of subpopulations which could not have been detected by using conventional methods.

Animals↗

PubMed1991

Mutagenic contaminants in synthetic peptides obtained by an azide coupling.

Hormone-like peptides are, almost by definition, not mutagenic. It was, therefore, unusual to find that some batches of peptides synthesized by azide coupling were mutagenic in the Ames test. One of these peptides, eledoisin, showed mutagenic activity particularly in Salmonella typhimurium TA 1535 without metabolic activation. This activity was independent of the peptide purity determined by HPLC and a dose response relationship was observed at concentrations over the solubility limit of the peptide in the assay medium. We therefore suggested that the mutagenic effect might be due to the presence of chemically undetectable, water-soluble impurities, which could be removed by counter-current distribution. If, however, the same final coupling was carried out by the mixed anhydride procedure, no mutagenic activity was observed. Consequently, we considered that the mutagenicity detected was due to traces of hydrazoic acid salts arising during azide formation in the coupling step. In fact only the product of the coupling reaction between the pivotal intermediates was mutagenic.

Amino Acid Sequence↗

PubMed1999

In vivo survival of selected murine carrier red blood cells after separation by density gradients or aqueous polymer two-phase systems.

In order to explore possibilities of using erythrocytes as carrier systems for delivery of pharmacological agents, we have studied the in vivo survival of murine carrier red blood cell populations enriched in young or old cells. Hypotonic-isotonic dialysis has been used to modify the cells as carrier systems and Percoll/albumin density gradients or counter-current distribution in aqueous polymer two-phase systems to separate them according to age. Hypotonic-isotonic dialysis produces a decrease in the red blood cell populations in vivo survival rate (from 9.5 to 7.8 days). Among the cells modified as carriers, the enriched young red blood cell populations show a higher in vivo survival (half-life 6.5-7.4 days) than populations made up of predominantly old red blood cells (half-life 4.7-6.2 days). Half-life of young or old circulating red blood cells was approximately one day longer when these cells were separated by counter-current distribution rather than by Percoll density gradients. Based on these results, hypotonic-isotonic dialysis of whole and enriched young or old red blood cell populations, with higher or lower survival rates, can be considered as a useful tool for modification of these cells as carriers. The final outcome of such changes can be translated into better control of plasma drug delivery during therapy.

Animals↗

PubMed2004

Fingerprinting of Salvia miltiorrhiza Bunge by non-aqueous capillary electrophoresis compared with high-speed counter-current chromatography.

The component of the traditional Chinese medicine (TCM) can be influenced by soil, climate, and growth stage, and fingerprint is an important means in its quality control. Our previous studies showed that high-speed counter-current chromatography (HSCCC) was helpful in the development of fingerprint of TCM. Since the HSCCC method is new, it is necessary to compare it with conventional ones, such as high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and high-performance capillary electrophoresis (HPCE). Comparison with HPLC was conducted in our previous study. In this study, HSCCC was compared with non-aqueous capillary electrophoresis (NACE). With NACE, seven stable components were separated within 55 min, respectively, from three crude samples of Salvia miltiorrhiza Bunge from different growth locations. In HSCCC separation, 12 components were separated, respectively, with good correspondence and precision within 13 h. Both NACE and HSCCC were effective in showing whole concentration distribution of all kinds of constituents. Principles of these two methods were very different, which led to different elution sequences and relative contents of peaks. HSCCC showed better performance in analysis of tanshinones, which made its fingerprint containing more chemical information than that of NACE. It was further proven that HSCCC could be a feasible and cost-effective method in the development of the fingerprint of TCM.

Countercurrent Distribution↗

PubMed1985

Demonstration of two erythrocyte populations in young chickens by counter-current distribution of 59Fe-labelled cells in dextran-poly(ethylene glycol) two-phase systems.

The technique of fractionating cells by a counter-current distribution procedure in dextran-poly(ethylene glycol) biphasic systems was modified by increasing the settling time and decreasing the top/bottom two-phase volume ratio. Two sub-populations of 59Fe-labelled erythrocytes, namely definitive medullar cells and primitive embryonic cells, were present in the blood of young chicks until about day 8 of age. The primitive cells were progressively replaced by definitive red cells as age increased.

Aging↗

PubMed1982

Microanalytical partition of rat-liver homogenates by poly(ethylene glycol)-dextran counter-current distribution.

1. The principal organelles from rat liver homogenates were fractionated by counter-current partition between solutions of dextran T50 and poly(ethylene glycol) 6000 in 0.25 M sucrose containing 10 mM sodium phosphate/phosphoric acid buffer, pH 7.4. 2. A small-volume (1.3-ml sample well)counter-current partition apparatus capable of 17 transfers, in which separation of the phases was enhanced by low-speed centrifugation, was used. 3. Variations in distribution of organelles was observed with increasing concentration of dextran and/or poly(ethylene glycol). At a constant polymer composition of 5.0% (w/w) dextran and 4.0% (w/w) poly(ethylene glycol), increasing concentrations of sodium chloride (2.5 mM, 5 mM and 10 mM) also altered the partition of the organelles. 4. Useful analytical resolution of the various organelles was achieved. In particular, separation of endoplasmic reticulum from plasma membrane components was obtained.

Animals↗

PubMed1976

Isolation and identification of a sulfur-containing metabolite of spironolactone from human urine.

In the urine of normal subjects who were given an oral dose of 500 mg spironolactone (3-(3-oxo-7alpha-acetylthio-17beta-hydroxy-4-androsten-17alpha-yl)-propionic acid gamma-lactone; Aldactone) together with 100uCi H-20, 21 spironolactone, a so far unknown major metabolite has been detected by thin layer chromatography. The metabolite then could be isolated by means of counter-current-distribution. According to masspectral and magnetic resonance data, the metabolite has been assigned the structure of 3-(3-oxo-7alpha-methyl sulfonyl-6beta, 17beta-dihydroxy-4-androsten-17alpha-yl)-propionic acid gamma-lactone. By oxidation of the corresponding methylsulfinyl compound - another already known metabolite of spironolactone - with m-chloroperbenzoic acid, a compound has been isolated which proved to be identical with the new metabolite according to TLC, MS and NMR.

Countercurrent Distribution↗

PubMed1985

Affinity partitioning and centrifugal counter-current distribution of membrane-bound opiate receptors using naloxone-poly(ethylene glycol).

Crude synaptic membranes isolated from calf brain cortex were subjected to an aqueous two-phase system and the partition of the various membrane constituents and activities between the phases were studied. These constituents were phosphate, cholesterol and protein. The activities measured were acetyl-cholinesterase, succinate dehydrogenase, 2',3'-cyclicnucleotide-3'-phosphohydrolase and stereospecific opiate-binding. The successful fractionation of the membranes was achieved by the use of an aqueous two-phase system in a counter-current distribution process. A ligand bound to poly(ethylene glycol) with an affinity for opiate receptors was synthesized by reacting 6-aminonaloxone with tresylpoly(ethylene glycol). The ligand-polymer was used to extract membrane-bound opiate receptors into the upper, poly(ethylene glycol)-rich phase. This use of affinity partitioning resulted in membrane fractions with a 3-4 fold higher ability to bind stereospecifically etorphine than the original preparations of synaptic membranes.

Animals↗

PubMed1973

Partition of Salmonella typhimurium in a two-polymer acqueous phase system in relation to liability to phagocytosis.

Analysis of the partition in a two-polymer phase system of Salmonella typhimurium R mutants with different susceptibility to phagocytosis distinguished between the mutants. Close to 80% of the R mutants least resistant to phagocytosis accumulated in the dextran-rich phase, whereas more phagocytosis-resistant mutants showed higher affinity for the interface and the polyethylene glycol-rich top phase. By growing the uridine diphosphate (pyro)-gal-4-epimeraseless mutant LT2-M1 in the presence of d-galactose for different periods of time, two well-defined peaks were obtained in counter-current distribution analysis with an aqueous two-polymer phase system revealing a heterogeneous population in the culture. One peak was located at a site characteristic of R bacteria, the other at the site of S bacteria. As growth proceeded, more bacteria transferred from the R-type to the S-type peak. Within each peak, a gradual increase in resistance to phagocytosis by rabbit polymorphonuclear cells occurred with increasing length of growth in d-galactose.

Countercurrent Distribution↗

PubMed2005

Enantiomer separation by counter-current chromatography. Optimisation and drawbacks in the use of L-proline derivatives as chiral selectors.

Several L-proline and (4R)-hydroxy-L-proline derivatives were evaluated as chiral selectors (CSs) in the separation of enantiomers by counter-current chromatography (CCC). A variety of biphasic solvent systems, all of organic/aqueous nature, were tested in order to determine the appropriate distribution for CSs and racemates (N-(3,5-dinitrobenzoyl)-(+/-)-leucine and (+/-)-ketoprofen). Successful separations of DNB-(+/-)-leucine in analogous experimental conditions allow the comparative study of the enantioselectivity displayed by the considered CSs. The low solubility of certain CSs limits their applicability for preparative purposes even for improved enantioselectivity. The effect that the nature and pH of the buffer solutions used as a component of the solvent system have on the separation was also studied.

Buffers↗

PubMed1983

Association of rabbit muscle glycolytic enzymes with filamentous actin. A counter-current distribution study at high ionic strength.

The association between purified glycolytic enzymes and filamentous actin from rabbit muscle has been studied by counter-current distribution. The co-distribution of a glycolytic enzyme and filamentous actin leads to a significant change in the counter-current distribution profile of the enzyme whereas that of actin is unaffected. The changes in the distribution profiles clearly demonstrated that all glycolytic enzymes studied, though to different extents, bind to filamentous actin. The aqueous two-phase system used for the studies contained dextran, poly(ethyleneglycol) and 150 millimolal potassium phosphate buffer, pH 7.0. Since the ionic strength of the two-phase system is determined mainly by the buffer, the glycolytic enzymes are evidently able to associate with filamentous actin, at least in the presence of neutral polymers, at ionic strengths comparable to or higher than those assumed to prevail in vivo.

Actins↗

PubMed1986

Fractionation of bone-marrow cells by counter-current distribution in aqueous polymer two-phase systems. Relation between settling time and the efficiency of separation.

The fractionation of heterogeneous populations of rat and human bone-marrow cells has been studied by counter-current distribution in a charged 5% dextran-4% poly(ethylene glycol) two-phase system. The subfractionation into two broad populations has been achieved at a low top/bottom phase volume ratio by increasing (up to 20 min) the settling time allowed for the phases to be separated after each mixing step. No effect of this parameter on a homogeneous population of erythrocytes has been observed. However, heterogeneous cell separations can be improved by exploiting different phase settling times.

Animals↗