PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Countercurrent Distribution”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 433 records · Page 24Linked to original sources
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↗
PubMed1993

Separation of bovine X and Y sperm based on surface differences.

Aqueous two-phase partition involving thin-layer counter current distribution (TLCCD) has been used to assess surface heterogeneity of ejaculated bovine sperm. When partitioned in charge-insensitive aqueous two-phase systems, which detect non-charge associated surface properties, the sperm fractionates into two distinct populations. Using a Y-chromosome-specific DNA marker, it has been shown that one of these populations is enriched in Y chromosome bearing sperm. However, this population is not pure--it consists of 80% Y sperm, with the other 20% being X sperm. All the sperm in the original population that had begun to undergo the acrosome reaction were separated into this same peak; the sex chromosome composition of these sperm is unknown. Since the aqueous partition of sperm is based on surface properties these results suggest that two populations of Y sperm exist that have different surface characteristics.

Animals↗
PubMed1996

Heterogeneity of hypotonically loaded rat erythrocyte populations as detected by counter-current distribution in aqueous polymer two-phase systems.

Carrier rat erythrocytes loaded with exogenous substances ([125I] carbonic anhydrase) by hypotonic-isotonic dialysis become heterogeneous cell populations that can be fractionated using the counter-current distribution (CCD) technique. Two well-defined low- and high-partition ratio, G, subpopulations are obtained in charge-sensitive dextran-polyethylene glycol two-phase systems. The low-G subpopulation, which contains the most fragile and surface-altered cells, as deduced from their osmotic fragility curves and partition behaviour, respectively, presents a high amount of exogenous substance incorporated (134.6 cpm/10(6) cells). The high-G subpopulation, that contains cells similar to the control or isotonically dialyzed cells presents a lower amount of exogenous substance incorporated (69.8 cpm/10(6) cells). Cells in this high-G subpopulation seem to be fractionated, like the controls, according to ageing as suggested by the decline of the pyruvate kinase specific activity from the left- to the right-hand side of the CCD profile.

Animals↗
PubMed1996

Surface properties of irreversibly sickled cells differ from those of the bulk of sickle cells. Studies by partitioning in aqueous phase systems.

Counter-current distribution (CCD) of red blood cells (RBC) from individuals with homozygous sickle cell (HbSS) disease in a charge-sensitive aqueous dextran-poly(ethylene glycol) phase system, which fractionates cells on the basis of surface properties, indicates that the percentage of irreversibly sickled cells (ISC) increases and the percentage of reticulocytes decreases with increasing cell partition ratios. The high partition ratios of ISC correspond to those of older RBC when RBC from normal individuals are subjected to CCD. Our results thus indicate that ISC differ in surface properties from those of the bulk of sickle RBC (including reticulocytes) in the population and that the difference is, most likely, charge-related. While the question as to whether ISC are indeed old cells has not yet been unequivocally answered, this view finds support in the fact that the independent parameters of ISC surface properties, as reflected by partition ratios, and densities correlate as they do in older RBC from normal individuals.

Cell Separation↗
PubMed1989

Partition coefficients of low-molecular-weight volatile chemicals in various liquids and tissues.

Partition coefficients are required for developing physiologically based pharmacokinetic models used to assess the uptake, distribution, tabolism, and elimination of volatile chemicals in mammals. A gas-phase vial equilibration technique is presented for determining the liquid:air and tissue:air partition coefficients for low-molecular-weight volatile chemicals. This technique was developed from two previously described medium:air methods, relied solely on measurement of chemical concentration in the gas phase, and, compared to earlier work, extends the range of chemicals and tissues examined. Partition coefficients were determined with 0.9% saline, olive oil, and blood, liver, muscle, and fat tissues from rats for 55 compounds. Human blood:air coefficients were determined for 36 compounds and several blood:air values were also determined in the mouse and for one compound in the hamster. An approach is described for predicting the tissue solubilities of untested compounds based on oil:air and saline:air coefficients using regression analyses. A similar approach is used to model fat:air coefficients in terms of oil:air values and to model human blood: air coefficients in terms of rat blood:air coefficients.

Adipose Tissue↗
PubMed2001

Sperm washing method alters the ability of seminal plasma proteins to revert the cold-shock damage on ram sperm membrane.

Prolonged exposure of spermatozoa to seminal plasma (SP) has been found to have adverse effects on sperm function. Therefore, the separation of mammalian spermatozoa from SP is a practice routinely used in the laboratory and in assisted reproductive technology application. We have previously shown that adsorption of seminal plasma proteins (SPP) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we have compared the influence of two different semen washing methods, a dextran/swim-up or a filtration procedure, on the SPP adsorption to the sperm surface, and on their ability to recover membrane integrity of cold-shocked sperm. Seminal plasma proteins were added to cold-shocked sperm samples obtained by both washing procedures. Adsorption of proteins to the sperm membrane surface was assessed by centrifugal counter-current distribution (CCCD) in an aqueous two-phase system, as well as membrane integrity being determined by fluorescence markers. The percentage of reversion of cold-shock effect in the sample containing plasma proteins with respect to the control sample was determined by assessing the percentage of membrane-intact spermatozoa, i.e. propidium iodide-negative. The addition of 700 microg of SPP to the swim-up damaged samples caused a 32% reversion, whereas the reversal percentage found with the same amount of SPP on filtered damaged samples was 10% (p < 0.05). Likewise, the loss of heterogeneity and the decrease in viability after the cold-shock revealed by CCCD analysis were greatly reversed by the addition of SPP to sperm samples obtained by swim-up. However, this restorative effect was again much lower when SPP were added to the cold-shocked filtered sample. These results strongly suggest that the washing method influences the ability of SPP to recover membrane integrity of cold-shocked sperm. Separation of spermatozoa by swim-up accounts for higher viability recovery than that obtained by filtration washing.

Animals↗
PubMed1980

The seminal excretion, plasma elimination, tissue distribution and metabolism of naltrexone in the rabbit.

The pharmacokinetics, tissue distribution and metabolism of naltrexone were studied in male New Zealand White rabbits. After an i.v. bolus, the plasma half-life of naltrexone between 30 min and 3 hr was 55 +/- 5 min and 53 +/- 3 min for 1 and 5 mg/kg doses of naltrexone . HCl, respectively. The drug concentration in the semen reached a maximum value between 15 and 30 min after the injection. At 120 min, the semen/plasma drug concentration ratio was 14 and 11 for the 1 and 5 mg/kg doses, respectively. Three minutes after injection, 95% of the drug had left the plasma. After 5 min, the conjugate levels exceeded the free drug levels in the plasma suggesting rapid glucuronidation of the drug. The concentrations of naltrexone and 6-beta-naltrexol were measured in different tissues 90 min after injection. Most of the tissues had drug concentrations which exceeded the concurrent plasma concentration. The highest concentrations were observed in the submaxillary gland. Relatively high amounts of 6-beta-naltrexol were found in the brain, fat, spleen, heart, testis, kidney and urine. The principle urinary metabolite was the glucuronide of naltrexone. Minor metabolites identified in urine treated with Glusulase were 6-beta-naltrexol and N-dealkylated naltrexone.

Animals↗
PubMed1996

Reversion of thermic-shock effect on ram spermatozoa by adsorption of seminal plasma proteins revealed by partition in aqueous two-phase systems.

Centrifugal counter-current distribution (CCCD) in a dextran, Ficoll, poly(ethylene glycol) two-phase system was used to study the effect of seminal plasma proteins on the partition behaviour of ram spermatozoa exposed to thermal shock. Ram spermatozoa freed from seminal plasma by a 'swim-up' procedure were submitted to thermal shock and fractionated by CCCD. Cell viability decreased from 68% to 18% after the treatment, showing a slight displacement of the cells from the right (where a higher enrichment of live cells is found) to the centre of the profile. A change of the distribution profile was shown in the presence of either ram or bull seminal plasma. Bull seminal plasma was able to move the profile to the right, whereas ram seminal plasma increased the proportion of cells with enhanced affinity for the lower dextran-rich phase. Plasma proteins isolated from both seminal plasmas moved the profile to the right. In addition, cell viability rose to 48% after the CCCD run in the presence of ram plasma proteins. This restoring effect was lost when ram plasma proteins were thermally denatured. Bovine serum albumin was not only unable to move the profile to the right but even promoted displacement of the profile to the left. This negative effect was also observed when proteins from bull seminal plasma were in the presence of protein-free ram seminal plasma. However, proteins isolated from ram seminal plasma still restored the profile in the presence of bull seminal plasma freed from proteins. The results presented here strongly suggest that seminal plasma proteins are adsorbed by a spermatozoal surface previously exposed to thermic shock. These proteins would exert a highly specific protective effect on ram spermatozoa. In addition, in the ram seminal plasma there must be some factor which avoids this adsorption.

Adsorption↗
PubMed1988

Partitioning behavior of erythrocytes in aqueous two-phase systems containing hydroxypropyl starch and polyethylene glycol.

The partitioning behavior of erythrocytes in Reppal PES 200 (a hydroxypropyl starch produced by Reppe Glykos AB, Växjö, Sweden)-polyethylene glycol (PEG) and in dextran (Dx)-PEG aqueous phase systems made isotonic with phosphate is similar in a number of ways: (i) There is a correlation between the relative electrophoretic mobilities and partition ratios, P, of red blood cells from different species; (ii) The cell P is reduced when, at constant polymer concentrations, phosphate is systematically replaced by sodium chloride (with the total concentration isotonic); (iii) The cell P is increased with reduced polymer concentrations (decreased interfacial tensions); (iv) Treatment of erythrocytes with neuraminidase results in a reduced P value; (v) Rat red cells of different ages can be fractionated by counter-current distribution; and (vi) Differences between red blood cells from genetically distinct rats or between humans can be detected. Aquaphase (a hydroxypropyl starch marketed by Perstorp AB, Lund, Sweden) has been tested as in ii-iv above with analogous results. The partitioning behavior of erythrocytes in PES-PEG and Dx-PEG aqueous phase systems containing sodium chloride differs in a number of ways: (vii) The correlation, apparent in Dx-PEG systems, between the P value of red blood cells from different species and the ratio of their membrane poly- to monounsaturated fatty acids is absent in PES-PEG systems. It is replaced by a correlation as in i; (viii) The increase in P value in Dx-PEG observed from red blood cells after treatment with neuraminidase is replaced by a decrease in P value in PES-PEG or Aquaphase-PEG systems. We conclude that PES (and Aquaphase) can be substitutes for dextran in cell partitioning studies when charge-sensitive phases are used (e.g., those containing phosphate) while separations based on properties reflected by Dx-PEG systems containing sodium chloride are not duplicated by PES-PEG (and probably not by Aquaphase-PEG). The hydroxypropyl starch-PEG systems containing sodium chloride, unlike the analogous Dx-PEG systems, have a significant electrostatic potential difference between the phases.

Aging↗
PubMed2001

Seasonal differences in ram seminal plasma revealed by partition in an aqueous two-phase system.

Seminal plasma plays an important role in maturation of spermatozoa through hormonal, enzymatic and surface-modifying events. We have previously shown that adsorption of seminal plasma proteins (SPPs) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we investigate the hypothesis that seasonal differences in seminal plasma could affect its ability to recover membrane integrity of cold-shocked sperm. The effect of seminal plasma proteins, obtained in breeding (bsSPPs) and non-breeding (nbsSPPs) season, on cold-shocked ram spermatozoa previously freed from seminal plasma, was analysed by centrifugal counter-current distribution (CCCD) in an aqueous two-phase system as well as membrane integrity determination by fluorescence markers. Cold-shock treatment greatly lowered cell viability in both breeding and non-breeding season spermatozoa. The cold-shocked sperm viability obtained was approximately 20%. The loss of heterogeneity and the decrease in viability revealed by CCCD analysis was reversed by the addition of increasing amounts of bsSPP, which induced restoration of the surface characteristics of viable-like spermatozoa, as well as an increase in the number of recovered viable sperm. However, this restoring effect was much lower when nbsSPPs were added, even in a sixfold higher concentration than used with bsSPPs. Incubation of cold-shocked cells with both kinds of proteins performed in both seasonal periods, showed that the recovering effect was related to the season when the plasma sample was obtained rather than to the semen season. The addition of bsSPPs to cold-shocked sperm accounted for a nearly 50% reversion for both studied breeding seasons. However, the reversion percentages obtained with nbsSPPs were significantly lower (P<0.05) than those found with bsSPPs in both studied seasonal periods. This different reversion capacity of bsSPPs and nbsSPPs was related to a different protein composition, as revealed by comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The bands of 20, 21, 24, 36 and 67 kDa of the bsSP sample profile decreased in winter-spring SP, and were even less intensely stained in summer SP. Densitometric analysis of the stained gel patterns allows automatic comparison among the separated bands, and revealed an important decrease in the content of several bands. The 21.5 kDa band showed the highest decrease, lowering to 14% in June-August plasma with respect to the value obtained in September-December plasma.

Animals↗
PubMed1997

Amphiphilic networks. X. Diffusion of glucose and insulin (and nondiffusion of albumin) through amphiphilic membranes.

Select semipermeable amphiphilic membranes have been prepared and their diffusional characteristics for glucose, insulin, and albumin investigated. The membranes were prepared by cast copolymerization of a hydrophilic monomer (i.e., N,N-dimethyl acrylamide, or N,N-dimethylaminoethyl methacrylate) with the hydrophobic crosslinker methacrylate-ditelechelic polyisobutylene. The products have sufficient mechanical properties for the fabrication of swollen membranes, sheets, tubes, etc. Membranes have been identified which allowed the rapid simultaneous countercurrent diffusion of glucose (Mn = 180 Da) and insulin (Mn = 5733 Da) but did not allow albumin (Mn approximately 60,000 Da) to pass. Evidently, the effective molecular weight cutoff point of these membranes is in the 6-60-KDa range.

Albumins↗
PubMed1996

Identification and characterization of various differentiative growth plate chondrocytes from porcine by countercurrent centrifugal elutriation.

Countercurrent centrifugal elutriation was used to separate growth plate chondrocytes from porcine basing on their differences in sizes and densities. Eighteen fractions of cells with different sizes and densities were obtained. The mean cellular volumes increased progressively in each of successive fractions, and that increase was associated with specific phenotypic changes, such as biochemical differences in DNA synthesis, proteoglycan synthesis, and activities of alkaline phosphatase. Three distinct chondrocyte subpopulations with their unique characteristics were identified among the elutriated fractions. The resting chondrocytes were found to be small in size and quiescent. The hypertrophic chondrocytes were found to be large in size and metabolically active both in alkaline phosphatase and in proteoglycan productions. The proliferative chondrocytes exhibited a high DNA synthesis rate, and their sizes were found to be between those of the resting and hypertrophic chondrocytes.

Alkaline Phosphatase↗
PubMedDate not supplied

Dye-affinity techniques for bioprocessing: recent developments.

Textile or triazine dyes play an important role as affinity ligands in protein purification. Each step of the protein purification protocol can be divided into three stages, partitioning between two phases, separation of these phases and recovery of the target protein from the enriched phase. Now developments in dye-affinity techniques are discussed emphasizing the innovations in all three stages of the protein purification process. Dye-affinity chromatography has become a routine step in protein purification. New dyes have been developed and used successfully in both traditional chromatographic mode and new modes like affinity precipitation, polymer aqueous two-phase partitioning or expanded bed chromatography. The specificity of dye techniques has been increased by both purposeful designing of new dyes and decreasing non-specific protein-dye interactions with polymer shielding. One can envisage further development and ramification of dye-affinity techniques in protein purification.

Affinity Labels↗