Countercurrent Distribution Research Articles

H-2053物質とルテオマイシンとの異同について-カウンターカレント法による

H-2053物質とルテオマイシンとの異同について-カウンターカレント法による

Hen egg yolk lipid fractions with antiatherogenic properties

Three different types of hen egg yolk, cage-free, organic and daily fresh, were tested for their antiatherogenic properties. Total lipids (TL) of all hen egg yolk samples were extracted by the method of Bligh and Dyer and further separated into total polar lipids (TPL) and total neutral lipids (TNL) by counter current distribution chromatography. TPL and TNL were further separated by preparative thin-layer chromatography (TLC). TL, TPL, TNL and the obtained polar and neutral lipid fractions after TLC separation were tested to determine whether they induce platelet activation or inhibit platelet activating factor (PAF)-induced platelet activation. All three hen egg yolk TL samples possessed strong inhibitory activity against PAF-induced platelet activation that was mainly attributed to TPL, especially to PL fraction 4. Cage-free hen egg yolk exhibited the most potent anti-PAF activity in all lipid classes (TL, TPL and TNL). Thus hen egg yolk contains PAF inhibitors that reinforce their nutritional value in terms of protection against cardiovascular diseases, since PAF is a crucial inflammatory phospholipid mediator that is implicated in the mechanism of atherogenesis.

Practical non‐chromatography strategies for the potential separation of PEGylated RNase A conjugates

AbstractBACKGROUNDThe implementation of efficient processes for the development, recovery and purification of biological products is a main challenge for the bioengineering field. PEGylation can enhance the therapeutic properties of the protein, and often results in heterogeneous population of conjugated species and unmodified protein that presents a protein separations challenge. Non‐chromatography strategies, such as countercurrent distribution in aqueous two‐phase systems (CCD‐ATPS) and ultrafiltration (UF) for the fractionation of PEGylated RNase A conjugates represent attractive alternative worth of consideration.RESULTSIn this work the potential application of CCD‐ATPS for the separation of closely related proteins (i.e. mono‐PEGylated and di‐PEGylated RNase A) was performed under selected conditions. Process conditions involved PEG 8000 13% (w/w), phosphate 10.4% (w/w), at pH 7.0 and volume ratio of 1.0 resulted, after 30 transfers in CCD, in distinguishable peaks that can be attributed to the mono‐PEGylated protein (61%) and di‐PEGylated (58%) form of RNase A. Alternatively, an ultrafiltration‐based strategy resulted in the partial separation of mono‐PEGylated and di‐PEGylated protein, 38% and 60%, respectively, in the concentrate with a 30 kDa membrane. Using a 50 kDa membrane the mono‐PEGylated RNase A accumulates in the filtrate (43%) and the di‐PEGylated in the concentrate (45%).CONCLUSIONSThe results reported here can be considered for the separation of PEGylated proteins, as a first step in the implementation of a purification process. Copyright © 2012 Society of Chemical Industry

Centrifugal countercurrent chromatography to elucidate surface differences of adipose tissue-derived stem cells

The current methods of isolation of adipose tissue-derived stem cells result in a heterogeneous population that might interfere with their differentiation potential and makes it difficult to compare the results between different groups. Partition in aqueous two-phase systems is one of the few techniques that separate cells on the basis of surface properties, gentle enough to isolate fragile cell types in isotonic conditions without altering their structure, and can be easily scaled. In this study, stem cells isolated from human adipose tissue seeded and expanded in vitro were fractionated by using centrifugal countercurrent distribution in an aqueous two-phase system. The separated subpopulations revealed the high heterogeneity of adipose tissue-derived stem cell samples. Comparative partition analyses showed that aging induces a loss of heterogeneity, which is not due to a loss of cell viability associated to age. The phosphatidylserine externalization, an apoptotic feature, is the main factor in cell partition that results in a decreased hydrophobicity of the cell surface. This procedure may be suitable for separating adipose tissue-derived stem cell populations enriched in some functional and/or structural surface characteristics. The possibility of a very effective separation of different subpopulations in opposite phases would be an interesting development of the method.

New cholinesterase inhibiting bisbenzylisoquinoline alkaloids from Abuta grandifolia

The phytochemical study of the stem bark and wood of Abuta grandifolia (Mart.) Sandwith led to the identification of four bisbenzylisoquinoline alkaloids (BBIQs), namely (R,S)-2 N-norberbamunine (1), (R,R)-isochondodendrine (2), (S-S)-O4″-methyl, Nb-nor-O6′-demethyl-(+)-curine (3), and (S-S)-O4″-methyl, O6′-demethyl-(+)-curine (4), together with the aporphine alkaloid R-nornuciferine (5), all obtained by countercurrent distribution separation (CCD) and identified on the basis of their spectroscopic data. Alkaloids 3 and 4 were new. All the isolated compounds were tested for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. 1 was the most active against AChE, whereas 3 and 4 were the most potent against BChE. Interestingly, all tested alkaloids are more potent against BChE than against AChE. This selectivity of cholinesterase (ChE) inhibition could be important in order to speculate on their potential therapeutic relevance.

Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20°C or cooled down to 5°C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/PI. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V, and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5°C than at 20°C, which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature.

Aqueous two-phase countercurrent distribution for the separation of c-phycocyanin and allophycocyanin from Spirulina platensis

C-phycocyanin (C-PC) and allophycocyanin (APC) with similar molecular structures were separated, respectively from Spirulina platensis cell homogenate by single extraction and multi-stage countercurrent distribution (CCD) using an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and potassium phosphate (KPi). The partition coefficients of C-PC and APC were 10.64 and 0.57, respectively, and the extraction selectivity of C-PC was 18.67 from 0.5% (w/w) S. platensis crude extract by single extraction using PEG6000/KPi ATPS (pH 7.0) with 34% (w/w) tie line length (TLL). In ten-stage CCD under the same ATPS extraction condition with 2% (w/w) S. platensis crude extract, the purity of C-PC increased nearly twice and the recovery of APC increased more than nine-fold compared with single extraction. The results displayed that most C-PC (82.1%) followed the mobile phase was enriched in the top phases of the last three tubes, while more APC (41%) remained in the stationary phase was enriched in the bottom phases of the first three tubes in the ten-stage CCD. Hence, aqueous two-phase CCD technology provided an effective and low cost method for C-PC and APC separation from S. platensis cell homogenate directly.

Rauwolfia alkaloids; fractionation by countercurrent distribution.

Journal Article Rauwolfia Alkaloids: Fractionation by Countercurrent Distribution Get access R P Banerjee, R P Banerjee Department of Pharmacology, School of Tropical Medicine, Calcutta Search for other works by this author on: Oxford Academic Google Scholar M L Chatterjee, M L Chatterjee Department of Pharmacology, School of Tropical Medicine, Calcutta Search for other works by this author on: Oxford Academic Google Scholar H F Häusler H F Häusler WHO Professor of Pharmacology 1954–55, Head of the Pharmacological Institute, University of Graz, Austria Search for other works by this author on: Oxford Academic Google Scholar Journal of Pharmacy and Pharmacology, Volume 9, Issue 1, September 1957, Pages 120–127, https://doi.org/10.1111/j.2042-7158.1957.tb12264.x Published: 12 April 2011 Article history Received: 11 September 1956 Published: 12 April 2011

Counter-current separation of constituents of Digitalis purpurea.

Abstract Mixtures of some glycosides of Digitalis purpurea have been partitioned in a mixture of ethyl acetate, benzene and water by counter-current distribution. The “A” and “B” series of primary glycosides were separated from the corresponding secondary glycosides. The partition ratio of each of the glycosides has been determined and used to calculate the theoretical distribution curve: this closely resembled the experimentally determined curve. Deviation of the “experimental” from the “theoretical” curve for digitoxin has been attributed to the presence of gitoxin in the sample.

Controlled-cycle pulsed liquid–liquid chromatography. A modified version of Craig's counter-current distribution

A new liquid–liquid chromatography technique developed from a combination of controlled-cycle operation and a pulsed-mixing technique is suggested and validated. The controlled-cycle pulsed liquid–liquid chromatography (CPLC) system operates without involving a centrifuge and consists, of a series of multistage units, and a method for imparting pulsation motion to the liquids inside the units (the pulsation cycle). This chromatography technique can be considered as an improved continuous form of Craig's counter-current distribution method, or, alternatively, as a form of droplet chromatography with the cycling mode of operation. The theoretical model has been designed to account for the effects of the basic parameters influencing the CPLC operation. The theoretical model's suitability was proved by direct comparison between the experimental and model responses. The CPLC devices containing 1, 2, 4 and 5 multistage columns (each column was divided into 26 stages) have been designed, fabricated and tested; experiments were conducted to test the chromatographic behavior of organic (monocarboxylic) and mineral acids. The mass transfer rate in the stages depends on the nature of both—phase and sample systems: the highest values were achieved in experiments with acetic acid by using the octane/water biphasic system, where an equilibrium concentration distribution between stationary and mobile phases in the stages was attained. The results obtained demonstrated the potential of the new technique for preparative and industrial scale separations.

Irritant and co-carcinogenic diterpene esters from the latex of Euphorbia cauducifolia L.

Ten (1–10) irritant and mild co-carcinogenic diterpene esters were isolated from the latex of Euphorbia cauducifolia L. using bioassay-guided countercurrent distribution and other chromatographic techniques. The isolated compounds were characterized on the basis of spectroscopic results and mass measurements. As an outcome, the ingenane-type esters were established with the following structures: 3-O-angeloyl-17-O-palmatoylingenol (1), 3-O-palmatoyl-5-O-angeloylingenol (2), 5-O-angeloyl-17-O-palmatoylingenol (3), 3-O-angeloyl-5-O-palmatoylingenol (4), 17-O-(2Z,4E,6Z)-2,4,6-tetradecatrienoyl-20-O-palmatoylingenol (5), 5-O-angeloyl-17-O-benzoylingenol (6), 5-O-angeloyl-17,20-diacetoxyingenol (7), 3-O-angeloyl-17-O-benzoyl-20-acetoxyingenol (8), 3-acetoxy-5-O-angeloyl-17-O-benzoylingenol (9), and 5-O-angeloyl-3,17,20-triacetoxyingenol (10). Their biological screening revealed that they are moderate irritants, and low to moderate tumor promoters compared to TPA, but hardly showed any solitary carcinogenic activity. The isolated esters represent new compounds and were not reported before from any source.

High pre-freezing dilution improves post-thaw function of ram spermatozoa

Despite considerable cryobiology research there is no industry standard for the concentration to which ram spermatozoa should be diluted before freezing. Ram semen is highly concentrated and often frozen at a high sperm concentration, necessitating the use of small laparoscopic insemination doses. The aim of this paper was to ascertain the effect of dilution on the integrity of frozen-thawed ram spermatozoa. In the first experiment, spermatozoa were extended with a Tris-buffered diluent before freezing or after thawing to yield a final sperm concentration of 20 × 10 6/ml, or were not diluted. Motility characteristics, viability and acrosome integrity of spermatozoa were analysed over a 6 h incubation period at 37 °C. In the second experiment, spermatozoa were either diluted before freezing, subjected to sex-sorting or not diluted before freezing. Thawed spermatozoa were separated into sub-populations using centrifugal counter-current distribution (CCCD) and the profile of partition and functional integrity (viability, chlortetracycline status and Annexin-V binding) in the sub-populations assessed. Dilution before freezing significantly improved post-thaw viability, acrosome integrity and total motility whereas dilution post-thaw decreased viability and motility of spermatozoa. Sperm heterogeneity, as assessed by CCCD profile, was not different for control, diluted and sex-sorted spermatozoa. Analysis of CCCD sub-populations showed the proportion of functional cells (displaying the F-Pattern or no PS translocation) was similar for all sperm types. The results show that ram spermatozoa retain normal function at higher pre-freeze dilution rates than are commonly used in the sheep industry. The application of these findings would result in more practicable and functional artificial insemination doses.

Letter to the Editor

Letter to the Editor

Universal counter-current chromatography modelling based on counter-current distribution

There is clearly a need for a model which is versatile enough to take into account the numerous operating modes and pump out procedures that can be used with counter-current chromatography (CCC). This paper will describe a universal model for counter-current chromatography based on counter-current distribution. The model is validated with real separations from the literature and against established CCC partition theory. This universal model is proven to give good results for isocratic flow modes, as well as for co-current CCC and dual flow CCC, and will likely also give good results for other modes such as intermittent CCC.

Fragmentation and separation analysis of the photosynthetic membrane from spinach

Membrane vesicles, originating from grana, grana core (appressed grana regions), grana margins and stroma lamellae/end membranes, were analysed by counter current distribution (CCD) using aqueous dextran-polyethylene glycol two-phase systems. Each vesicle population gave rise to distinct peaks in the CCD diagram representing different vesicle subpopulations. The grana vesicles and grana core vesicles each separated into 3 different subpopulations having different chlorophyll a/b ratios and PSI/PSII ratios. Two of the grana core subpopulations had a chlorophyll a/b ratio of 2.0 and PSI/PSII ratio of 0.10 and are among the most PSII enriched thylakoid vesicle preparation obtained so far by a non detergent method. The margin vesicles separated into 3 different populations, with about the same chlorophyll a/b ratios, but different fluorescence emission spectra. The stroma lamellae/end membrane vesicles separated into 4 subpopulations. Plastoglobules, connected to membrane vesicles, were highly enriched in 2 of these subpopulations and it is proposed that these 2 subpopulations originate from stroma lamellae while the 2 others originate from end membranes. Fragmentation and separation analysis shows that the margins of grana constitute a distinct domain of the thylakoid and also allows the estimation of the chlorophyll antenna sizes of PSI and PSII in different thylakoid domains.

An Editing Activity That Prevents Mistranslation and Connection to Disease

I believe that the genetic code was the greatest discovery of the 20th century (Fig. 1). The code was worked out during the period that I was a student: a medical student at Tufts University (I dropped out after a year), a graduate student at MIT and Cornell (with Gordon Hammes), and a postdoctoral student at Stanford University (with Paul Flory). My research as a student was centered on theoretical and experimental physical chemistry, as applied to biological systems. With Hammes, I had done a spectroscopic and thermodynamic analysis of ribonuclease and its interaction with one of its ligands. I also had done both experimental and theoretical analyses of kinetic relaxation spectroscopy, inspired in part by the pioneering work of Eigen, who himself had been a mentor of Hammes. (Eigen gave lectures at Cornell that I attended, and we spent time in conversation as well. In more recent years, we have seen each other on a regular basis and continued our conversations.) With Flory at Stanford, I worked on statistical mechanics of polypeptides. All of that graduate and postdoctoral work was exciting, particularly the mathematical side that I especially loved because of its elegance, rigor, and abstraction. However, while this sort of mental activity was going on, I kept thinking about the newly discovered genetic code and aminoacyl-tRNA synthetases.

Seymour Benzer 1921–2007

Seymour Benzer 1921–2007

Practical approach to protein recovery by countercurrent distribution in aqueous two‐phase systems

AbstractBACKGROUND: The development of efficient processes that will rapidly deliver new biological products to the market is a key goal in biochemical engineering. The establishment of purification processes that permit the recovery of closely related products is essential. The recovery and purification of these types of products is not a trivial task. The current approach exploiting chromatography techniques demands practical alternatives to address the technical and economic constraints. The potential application of countercurrent distribution in aqueous two‐phase systems (CCD‐ATPS) for the purification of related proteins is an alternative of great potential worth considering.RESULTS: The evaluation of the potential application of CCD‐ATPS for the recovery of proteins provided conditions under which three model proteins (i.e. bovine serum albumin (BSA), ovalbumin and α‐lactoalbumin) of similar isoelectric point but different molecular size can be separated. Process conditions PEG 1000 13% (w/w), phosphate 14% (w/w) at pH 7.0 and volume ratio of 1.0 resulted, after 32 transfers in CCD, in three distinguishable peaks that corresponded to each of the selected proteins.CONCLUSIONS: The findings reported here demonstrate a practical strategy that serves as a first step to establishing initial conditions to further exploit the separation of closely related proteins. Copyright © 2007 Society of Chemical Industry

Modelling counter-current and dual counter-current chromatography using longitudinal mixing cell and eluting counter-current distribution models

Two well known approaches are considered to analyze the processes of counter-current and dual counter-current chromatography: the longitudinal mixing cell model and the Craig's counter-current distribution model. The cell model represents perfectly mixed, equally sized cells in series. The number of cells characterizes the rates of longitudinal mixing in the stationary and mobile phases. In the eluting counter-current distribution (CCD) model, the CCC process is considered as a continuous form of Craig's counter-current distribution. For a cascade of equilibrium stages theoretical elution profiles of the CCC process by using the CCD and cell model approaches have been compared. It is shown that in general, distribution functions of the CCD and cell models differ. It is established that the distribution of a solute between two solvent phases in the dual CCC process is determined by the extraction factor c, the total number of equilibrium stages n and the position of the sample inlet m by the equation Q x = (1 − c m)/(1 − c n + 1 ) with c = F 2 K D/ F 1 ( K D, F 1, F 2 and Q x are the distribution constant, the phase flow-rates and the portion of solute eluted by the first phase, respectively).

Analysis of new counter-current chromatography operating modes

Some novel cyclic operating modes of counter-current chromatography have been suggested. Chromatographic separation processes with two zones of different partition coefficients and with extraction and scrubbing (washing) zones are mathematically described using the eluting counter-current distribution approach. Two possible cyclic modes of dual counter-current chromatography operation, with simultaneous and alternate transfers of the phases, are analyzed. It is shown that the operating mode with the alternate transfers of the phases provides considerably higher separation efficiency than the mode with simultaneous transfers of both phases.