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Results for “Countercurrent Distribution”

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At least 271 records · Page 15Linked to original sources

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↗

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↗

PubMed1982

[Separation of cells of the erythrocyte series of birds and mammals by distribution in biphasic polyethylene glycol-dextran systems].

Experimental conditions for the partition, in dextran (Dx) polyethylene glycol (PEG) biphasic systems, of circulating red cells, reticulocytes and bone marrow cells from adult rats, as well as of erythrocytes from chicken embryos, young animals and adult chickens, have been studied. Once the partition coefficient (K) has been calculated under optimal experimental conditions, a counter-current distribution (CCD) apparatus has been used for the separation of cells in those populations, and the influence of different factors on the distribution of cells has been analyzed. An increase in the distribution efficiency of heterogeneous cells (those from the chicken and bone marrow of rats) is achieved by increasing the sedimentation time during the CCD procedure. The position of cells (previously labelled with 59Fe) along the rotor of the CCD apparatus is related to cells age, and thus allowed us to apply the CCD procedure for the assay of some enzyme activities in the separated cells.

Animals↗

PubMed1989

The use of aqueous two-phase systems to concentrate and purify bovine leukemia virus outer envelope protein gp51.

Enzootic bovine leucosis is a chronic lymphoproliferative disease of cattle. The causative agent, bovine leukemia virus (BLV), is related to the human retroviruses HTLV-I and -II. The external env-protein of BLV, a glycoprotein of 51 kDa, carries neutralizing epitopes and should be an essential component in a vaccine against the virus. Problems have been encountered with the concentration and purification of intact virions of BLV and other retroviruses. During centrifugation procedures the external env-proteins are to a great extent detached and consequently poorly recovered with the virion particles. Therefore, other methods are sought to obtain a high yield of the external glycoproteins. The use of two-phase systems based on water soluble polymers is described for the extraction of BLV-gp51 from culture medium. Several polymer systems were tested and the results showed that some were attractive for large scale application. The classical combination dextran-polyethylene glycol gave promising results; a partition coefficient of about 0.02 was obtained for the distribution of the gp51 between the top and combined inter- and bottom phases. In a single extraction step it was possible to obtain 45% of the glycoprotein in a small volume bottom phase and at the same time about 15-fold purified. That should be compared with a recovery of less than 20% with the conventional centrifugation procedures. It is concluded that extraction in phase systems based on water soluble polymers is a methodology well suited for the concentration and purification of BLV-gp51.

Animals↗

PubMed1986

Effect of dextran- and poly(ethylene glycol)-bound procion yellow HE-3G on the partition of membranes from calf brain synaptosomes within an aqueous two-phase system.

Membranes obtained by lysis and Yeda-press treatment of synaptosomes (nerve endings) from calf brain cortex have been partitioned within the aqueous phases (and the interface between them) of a Ficoll-dextran-poly(ethylene glycol)-water two-phase system. By introducing the dye Procion yellow HE-3G in the upper phase, bound to poly(ethylene glycol), or in the lower phase, bound to dextran, the partition of the membranes could be strongly affected. The influence on the partition was more pronounced when the dye was bound to dextran. By using a number of two-phase systems in a counter-current distribution process, it was shown that the membrane preparation was inhomogeneous and that the fractions obtained differed in their contents of acetylcholinesterase, succinate dehydrogenase and ATPase. The affinity partitioning effect depended strongly on the concentration of polymer-bound dye. An optimum dye concentration was found when Procion yellow HE-3G was bound to poly(ethylene glycol). When the same dye was bound to dextran, the number of dye molecules per dextran molecule influenced the effectiveness of the extraction.

Acetylcholinesterase↗

PubMed1981

Binding of human carbonic anhydrase to human hemoglobin.

The ability of human carbonic anhydrases to interact with human CO-hemoglobin have been studied with the counter-current distribution technique in aqueous/aqueous biphasic systems. The experimental results show that human carbonic anhydrase II interacts with human CO-hemoglobin whereas human carbonic anhydrase I does not. THe interaction between CO-hemoglobin and carbonic anhydrase II was quantified using the theoretical model developed previously for one-to-one interacting systems. [Backman, L. and Shanbhag, V.P. (1979) J. Chromatogr. 171, 1-13]. The apparent association constant was estimated to be 4.1 x 10(5) l mol-1 at pH 8.0 and 21 degrees C for the association of carbonic anhydrase II and CO-hemoglobin.

Carbonic Anhydrases↗

PubMed1976

Isolation and chemical characterization of antitumor agents from plants.

This paper reviews methods which have evolved at the Research Triangle Institute during the last 15 years for the isolation and characterization of antitumor agents from plants. The isolation procedures stress mild, nonchemical methods. Solvent partition and the Craig Counter Current Distribution are used during the early stages of fractionation. Subsequent purification involves many types of chromatography: adsorption, partition, thin-layer, preparative thin layer, gel-exclusion, and medium- and high-pressure liquid chromatography. The pure compound is crystallized from a suitable solvent. The fractionation is monitored with in vitro and in vivo bioassays. Physical methods used for structure determination are: ultraviolet, infrared, and nuclear magnetic resonance spectrometry, and X-ray crystallography.

Alkaloids↗

PubMed1991

Changes in glycolytic enzyme activities in aging erythrocytes fractionated by counter-current distribution in aqueous polymer two-phase systems.

Human and rat erythrocytes were fractionated by counter-current distribution in charge-sensitive dextran/poly(ethylene glycol) two-phase systems. The specific activities of the key glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) declined along the distribution profiles, although the relative positions of the activity profiles were reversed in the two species. These enzymes maintained their normal response to specific regulatory effectors in all cell fractions. No variations were observed for phosphoglycerate kinase and bisphosphoglycerate mutase activities. Some correlations between enzyme activities (pyruvate kinase/hexokinase, pyruvate kinase/phosphofructokinase, pyruvate kinase/pyruvate kinase plus phosphoglycerate kinase, pyruvate kinase/bisphosphoglycerate mutase and phosphoglycerate kinase/bisphosphoglycerate mutase ratios) were studied in whole erythrocyte populations as well as in cell fractions. These results strongly support the fractionation of human erythrocytes according to cell age, as occurs with rat erythrocytes.

Animals↗

PubMed1980

Fingerprint analyses of plant and animal tissues with respect to the occurrence of foreign compounds.

Glass capillary chromatography can be used for the characterization of complex mixtures of plant and animal samples after clean-up by counter-current distribution. For good reproducibility of fingerprint patterns a special splitless injection system is used. This method prevents any loss at injection. In order to determine organochlorine substances also in trace amounts (a few pg) an electroncapture detector (ECD) was used. Each step in the analysis procedure was checked with the aid of radioactively labelled substances, e.g. the polar compound pentachlorophenol and the non-polar compound hexachlorobenzene. With the aid of capillary chromatography-mass spectrometry substances in complex mixtures in a concentration of 10 to 100 ng/microliters can be characterized. Suitable glass capillaries for these investigation were prepared in this laboratory. The results indicate that a standardization of the procedure is practicable.

Animals↗

PubMed1982

[Characteristics of the distribution in a 2-phase polymer system of L cells at different stages of culture growth].

By the method of counter-current distribution in two-phase polymer system Dextran-500/poly (ethylene)glycol-6000, surface properties of monolayer and suspension sublines of L cells were investigated in relation to the stage of culture growth. The cultivation conditions of both the cell types were identical. It has been shown that with the growth of cell population, the number of cells with lower partition coefficient increases in both the sublines, mostly in cells of monolayer subline. These data suggest more essential changes of surface properties of cells growing in monolayer. It is supposed that this can be associated with the inability of suspension cells to form adhesion contacts.

Animals↗

PubMedDate not supplied

Dermostatin A and B: chromatography, structural and configurational studies using HPLC, CCD, 13C (125 MHz) and 1H (500 MHz) NMR spectroscopy.

HPLC of crude Dermostatin indicated presence of three pairs of components. Hence, attempts were made to purify Dermostatin. Purification of crude Dermostatin has been carried out using column chromatography and counter current distribution methods. Each of these fractions were tested for activity. The major fraction which showed greater activity was taken for the preparation of Dermostatin nona-acetate. Structural characterisation of Dermostatin nona-acetate has been carried out using UV-visible spectroscopy in different solvents to obtain the characteristic spectrum of a carbonyl conjugated hexaene at room temperature. Structural and configurational studies of Dermostatin nona-acetate using 500 MHz 1H NMR and 125 MHz 13C NMR has been used in the assignment of various functional groups in Dermostatin A and B as well as to provide corroboration to the earlier structural elucidation.

Antifungal Agents↗

PubMed1973

Characterization of mutants of Salmonella typhimurium by counter-current distribution in an aqueous two-polymer phase system.

An aqueous, two-polymer phase system was employed in an attempt to separate and characterize a series of R mutants, derived from Salmonella typhimurium 395 MS, with differing lengths of lipopolysaccharide chains on their surfaces. R mutants with varying degrees of virulence and phagocytic resistance were unresolved in this system. However, the smooth MS bacteria were clearly separated from the rough cells and showed a high affinity for the polyethylene glycol-rich top phase. A uridine 5'-diphosphate-gal-4-epimeraseless mutant, phenotypically in R or S form depending on the growth medium, partitioned as the R mutants and S bacteria, respectively. These results demonstrate the great influence of long polysaccharide chains on the physicochemical properties of the cell surface.

Bacteriological Techniques↗

PubMed1981

Highly purified mitochondria from rat brain prepared by phase partition.

Mitochondria and synaptosomes from adult rat forebrain can easily be separated by counter-current distribution in an aqueous two phase system composed of Dextran T500 and poly(ethylene glycol) 4000. Both particles may also be separated by a batch procedure in which the same phase system is used. Electron micrographs and enzymatic activities show a high purity of the mitochondria obtained from the dextran-rich lower phase. Electron micrographs and enzymatic activities also show that intact synaptosomes can be obtained from the poly(ethylene glycol)-rich upper phase. The mitochondria purified by this method show good ADP/O ratios, respiratory control ratios, and state 3 rates. Synaptosomes showed a state 2-state 3 transition with no recuperation to state 4.

Acetylcholinesterase↗

PubMed1976

Separation of subchloroplast membrane particles by counter-current distribution.

Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity. The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique. Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks. The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicated that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae.

2,6-Dichloroindophenol↗

PubMed1985

Trichotoxin A40. Purification by counter-current distribution and sequencing of isolated fragments.

The isolation of the membrane-modifying polypeptide antibiotics from the mycelium of Trichoderma viride 5242 was optimized via extraction with dichloromethane and chromatography on Sephadex LH-20. The components trichotoxin A40 and A50 were separated from each other and purified by multiplicative counter-current distribution. The sequence of proteinase-resistant trichotoxin A40 was determined by combined gas chromatography and mass spectrometry of three isolated N-acetylated dodecapeptides and two N-prolylhexapeptides obtained after selective trifluoroacetolysis. Including amino acid exchanges due to natural microheterogeneity, the sequence is Ac-Aib-Gly(LAla)-Aib-LLeu-Aib-LGln-Aib-Aib-Aib(LAla )-LAla-Aib-Aib-LPro-LLeu -Aib-DIva(Aib)-LGlu-LValol. In contrast to the eicosapeptide alamethicin, trichotoxin A40 contains only 18 residues, with a higher proportion of alpha-aminoisobutyric acid (Aib), C-terminal L-valinol (Vol), one D-isovaline (Iva) and no proline at the N-terminal part.

Amino Acid Sequence↗

PubMed1976

Hydrophobic affinity partition of spinach chloroplasts in aqueous two-phase systems.

The surface properties of spinach chloroplasts, both of intact chloroplasts with surrounding envelope and broken chloroplasts consisting of the inner lamellar system, have been studied by partitioning them between two aqueous phases, especially using counter-current distribution technique. The two-phase system consists of poly(ethyleneglycol), dextran and water. The two polymers are enriched in opposite phases and by binding deoxycholate or palmitate to one of the polymers the affinity of chloroplasts for the corresponding phase is strongly enhanced. The partition of the two classes of chloroplasts, however, is not affected to the same degree and the affinity of the chloroplast envelope for deoxycholate and palmitate is stronger than that of the lamellar system. This has been correlated to the chemical composition of the two types of membranes. By studying the effect of salts on the partition it has been found that the lamellar system bears a larger number of negative charges as compared to the envelope of the intact chloroplast.

Binding Sites↗

PubMed1994

Revealing surface changes associated with maturation of ram spermatozoa by centrifugal counter-current distribution in an aqueous two-phase system.

Centrifugal counter-current distribution (CCCD) in an aqueous two-phase system was used to detect changes associated with maturation of ejaculated ram spermatozoa. Spermatozoa obtained from three successive ejaculates of rams maintained in abstinence for one, two and three days were fractionated by CCCD. The results show that these ejaculates are relatively enriched in a cell population which presents a very high enhanced affinity to the lower dextran-rich phase. This cell population is not associated with loss of acrosomal integrity. In addition, it tends to disappear with longer abstinence periods, or after successive ejaculations at the same abstinence period, strongly suggesting that it is composed of immature cells. Therefore, phase partitioning can detect surface changes accompanying sperm maturation and offers a new possibility for sperm quality analysis.

Animals↗