High Ionic Strength Media Research Articles

Effect of High Ionic Strength and Inhibitors of H,K-ATPase on the Ouabain-Sensitive K-p-Nitrophenylphosphatase Activity in the Sea Anemone Stichodactyla helianthus

The ouabain-sensitive, K-stimulated p-nitrophenyl phosphatase (K-pNPPase) activity, an associated activity of the Na,K-ATPase, was assayed in tentacles of the sea anemone Stichodactyla helianthus to investigate the possibility that the sea anemone Na,K-ATPase activity is an associated activity of an H,K-ATPase. Activity was maximal at pH 6.5–7.0, decreasing only slightly in acidic medium but falling abruptly in alkaline medium to 60% of maximum at pH 7.4. The pH of maximum activity was not remarkably altered in high ionic strength medium (560 mM choline chloride), but ouabain-sensitive K-pNPPase activity of both rat and sea anemone was strongly inhibited. Inhibitors of the gastric H,K-ATPase, 100 μM omeprazole and 10 μM SCH 28080, did not inhibit the ouabain-sensitive K-pNPPase activity. Activity of the sea anemone enzyme was inhibited by 10 μM ammonium vanadate, an inhibitor of P-type ATPases, and not by 2.5 mM sodium azide, an inhibitor of both F-type and V-type ATPases. Because the sea anemone K-pNPPase activity was previously found to be more sensitive to ouabain than the Na,K-ATPase activity, K+-ouabain antagonism was investigated and found to be relatively muted, whereas K+-Na+ competition was stronger than in the rat kidney.

Preparation of Neoglycoprotein from Carp Myofibrillar Protein by Maillard Reaction with Glucose: Biochemical Properties and Emulsifying Properties

Neoglycoprotein was prepared from carp myofibrillar protein by using the Maillard reaction with glucose, and its solubility in a high ionic strength medium, Ca-ATPase activity, and emulsifying properties were investigated. For reacting with glucose, the lyophilized protein mixed with glucose (the weight rate: 1:9) was incubated at 40 °C (for 24 h), 50 °C (for 6 h), or 60 °C (for 3 h) and 65% relative humidity. Fructosamine was produced during incubation, and available lysine content decreased simultaneously at all temperatures. In these conditions, the Maillard reaction occurring between a lysine residue and glucose was in the early stage. The glycosylated protein has high solubility in 0.5 M NaCl, and its emulsifying properties were superior to unglycosylated protein, although Ca-ATPase activity was completely lost. The regulation of the Maillard reaction in the early stage would be important for improvement of functional properties of fish myofibrillar protein. Keywords: Fish; myofibrillar protein; myo...

Mutational Analysis of a Plant Defensin from Radish (Raphanus sativus L.) Reveals Two Adjacent Sites Important for Antifungal Activity

Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI beta-turn connecting beta-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting beta-strand 1 and the alpha-helix and contiguous residues on the alpha-helix and beta-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca2+ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca2+ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca2+ uptake.

Comparison of the chelating ion exchange properties of dye coated cellulose and polystyrene substrates for the separation and determination of trace metals from aqueous media

The following paper describes the comparison of a number of dye impregnated polystyrene resins and dye coated cellulose for the chelating ion exchange of metal ions. Dye loadings, capacity factors, metal adsorption capacity and selectivities were measured. The cellulose substrate was found to have a strong affinity for copper but had a very low capacity. Of the polysterene resins, highly cross-linked macronets gave the highest metal adsorption capacities. For one of the resins, an example of a separation of a synthetic mixture of alkaline earth metals in high ionic strength media is described.

Human dentine as a hydrogel

The pores (tubules) of human dentine in 0.02-cm planoparallel sections of newly extracted permanent teeth were investigated. By the conventional scanning electron microscopy these pores appear empty, but by the newly developed scanning-probe microscopy the presence of a complex matrix could be established. By measuring the transport of neutral myoglobin by diffusion alone and diffusion + bulk flow, the area of dentine occupied by the matrix was calculated to be 1.9 ± 0.9% and 2.3 ± 0.5%, respectively. The hydraulic conductivity was surprisingly small, 1.35 ± 0.55 × 10 −7 ml/(s·cm 2 dentine) at a pressure difference of 0.1 kPa across a 1-cm thick section. This suggests a hydrogel with a relatively dense network, the width of meshes estimated at 2 × 30 nm. In line with this concept, enzymatic degradation of the organic matter increased the hydraulic conductivity 3000 times. By studying the transport of negatively charged myoglobin, the matrix was calculated to carry 18 mEq/1 of positive charges. Due to the consequent attraction of small, negative ions and thence of water, the pressure within the matrix would be about 1.33 kPa, a force which will act to immobilize the water in the channels. The concept of a hydrogel in the dentine tubules was also supported by the finding that shielding the charges with bathing media of high ionic strength reduced the hydraulic conductivity.

Selective and asymmetric molecular transport across electroporated cell membranes.

Transport of a divalent cation (Ca2+) and three DNA indicators [ethidium bromide (EB), propidium iodide (PI), and ethidium homodimer (EthD-1)] across electroporated membranes of several mammalian cell lines was found to be selective and asymmetrical. In low salt medium, Ca2+ and EB were preferentially transported across the anodefacing cell membrane while PI and EthD-1 predominately entered at the site facing the cathode. In high salt medium, the entry site for Ca2+ and EB was reversed to the cathode-facing hemisphere while it remained unchanged for PI and EthD-1. In all these experiments, the observed transport patterns remained unaffected whether the dyes (or ion) were present during or added after the electroporating pulse. The data suggest that asymmetric pores are created on both sides of the membrane facing the electrodes, with smaller pore size (but greater in number) on the anode side and larger pores (with a lower population) on the cathode side. Furthermore, the rate of resealing of the membrane pores is significantly enhanced in high ionic strength medium, thus affecting the entry site. The asymmetric transport pattern is neither caused by electrophoresis induced by the externally applied electric field nor due to one-sided membrane breakdown as previously believed.

Influence of exercise on the distribution of enzymes in trout white muscle and kinetic properties of AMP-deaminase from free and bound fractions

Effects of exercise on the distribution of phosphofructokinase (PFK), fructose-1,6-biphosphatase (FBPase), and AMP-deaminase between free and particulate-bound fractions was analyzed in white skeletal muscle of rainbow trout Oncorhynchus mykiss. With a widely used technique for the separation of free and bound enzyme fractions (homogenization in low ionic strength, high sucrose buffer), the data showed that the amount of bound PFK increased from 64 to 95% during burst swimming whereas other enzymes were unaffected. Since this data for AMP-deaminase contrasted with earlier reports, different methods of separating free and bound enzyme were evaluated. A clear effect of exercise on AMP-deaminase binding occurred when high ionic strength media (either KCl or KF) were used; in extraction media containing 150 mM KCl, the percent bound rose from 30% in controls to 97% after 1 min burst swimming. Exercise also produced stable changes to AMP-deaminase kinetic properties, including for the bound enzyme (compared with the free) a 2-fold higher Km AMP, a 3-fold higher Ki for inorganic phosphate, and a 60% increase in Ka ADP after 1 min burst exercise. The data suggest that AMP-deaminase in working skeletal muscle is subject to combined controls by allosteric effectors, post-translational modification, and distribution between free and bound states.

Role of electrostatic interactions in the adhesion of Pseudomonas fragi and Brochothrix thermosphacta to meat

Microbial contamination of meat during slaughter is unavoidable. The adhesion of two species of alterating flora, Pseudomonas fragi and Brochothrix thermosphacta, were studied. These strains adhered readily and instantaneously to lean and fat beef tissue. The adherent population of the two strains of P. fragi was larger than that of B. thermosphacta. Attachment was not affected by pH variation (5.8–7.2). Pseudomonas fragi attachment was time dependent between 1 and 10 min, unlike that of B. thermosphacta. To determine the influence of electrostatic interactions on bacterial adhesion to meat, we studied surface charges by measuring the electrophoretic mobility and bacterial attachment in relation to the ionic strength of the attachment medium (0.15 M and 0.0015 M phosphate-buffered saline (PBS)). Using the low ionic strength medium (0.0015 M PBS), electrostatic interactions were repulsive and reduced bacterial attachment to lean beef tissue, unlike that which was observed for fat tissue. Using the high ionic strength medium (0.15 M PBS), a medium similar to that of meat interstitial fluids, electrostatic interactions seem negligible. Thus, in future studies, the thermodynamic and specific interactions will be investigated.

Differential effects of polylysine on NADH‐linked electron transfer in corn root plasma membrane vesicles

Abstract The effects of polylysine on the electron transfer from NADH to cytochrome c and ferricyanide in corn root plasma membrane vesicles were investigated. The presence of polylysine decreased the reduction rate of cytochrome e but slightly stimulated the rate of ferricyanide reduction in a pH 7.5 medium of low ionic strength. An increase in ionic strength eliminated the effects of polylysine on ferricyanide reduction but not the inhibition of cytochrome c reduction. Kinetic analyses indicated that in high ionic strength medium, polylysine inhibition appeared to assume a competitive model suggesting its binding to cytochrome c reduction site. In low ionic strength medium, a complex inhibition was observed. A study on the inhibitory efficiency of different size polylysines suggested that the reduction site of cytochrome c may best fit a polylysine with molecular weight close to 25,000. The results support the notion that the electron transfer from NADH to these two acceptors may involve two separated e...

Platelet activating factor enhances the acrosome reaction, fertilization in vitro by subzonal sperm injection and resulting embryonic development in the rabbit.

This study was conducted to investigate the effect of platelet activating factor (PAF) on the acrosome reaction and fertilizing capacity of spermatozoa, and development of the resulting embryos in the rabbit. Rabbit spermatozoa were exposed to PAF, lyso-PAF, or high ionic strength medium (HIS) prior to subzonal sperm injection (SUZI) into 326 mature oocytes, or morphological assessment of the acrosome reaction. The rates of fertilization and blastocyst formation were compared among the three treatment groups. Acrosome reaction was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining and electron microscopy. PAF-treated spermatozoa fertilized the oocytes at a significantly higher rate (56.1%) than did lyso-PAF-(36.8%, P < 0.01) or HIS- (38.2%, P < 0.05) treated spermatozoa. The embryos produced by PAF-treated spermatozoa showed significantly higher blastocyst formation rates (34.0%) than lyso-PAF- (8.6%, P < 0.05) or HIS-(8.8%, P < 0.05) treated spermatozoa. FITC-PSA staining demonstrated a significantly higher incidence of acrosome reaction in PAF-treated spermatozoa (45.8%) than in lyso-PAF- (28.0%, P < 0.01) or HIS- (34.9%, P < 0.01) treated spermatozoa. Acrosome reaction of PAF-treated spermatozoa was also confirmed by electron microscopy. PAF treatment of spermatozoa enhances fertilizing capacity for SUZI possibly by augmenting the acrosome reaction. Enhanced embryonic development was also found in the oocytes fertilized by SUZI of PAF-treated spermatozoa.

Type II phospholipase A 2 in human gestational tissues: extractable immuno- and enzymatic activity in fetal membranes

In this study, we have established the presence of immunoreactive (ir) Type II PLA 2 in human amnion and choriodecidua obtained from women at term prior to the onset of labour. The content of irType II PLA 2 present in 1 M NaCl extracts of choriodecidua and amnion averaged 3.5 ± 3.1 and 10.6 ± 5.2 ng/ mg tissue protein ( n = 3), respectively. PLA 2, enzymatic activity present in the same tissues averaged 1.3 ± 0.2 and 1.9 ± 0.7 nmol phosphatidylethanolamine (PE) hydrolysed/mg tissue protein per h ( n = 3), respectively. To allow intra-patient comparison of the relative distribution in gestational tissues, irType II PLA 2 and PLA 2 enzymatic activity was also determined in placenta obtained from the same group of women, and averaged 26.0 ± 7.0 ng/mg tissue protein and 3.5 ± 1.0 nmol PE hydrolysed/mg protein per h ( n = 3), respectively. As has been previously reported for human placenta, the recovery of Type II PLA 2 and PLA 2 enzymatic activity from amnion and choriodecidua was increased between 16- and 25-fold when tissues were homogenized in high-ionic strength media (i.e., 10% (w/v) ammonium sulphate or 1 M NaCl) compared with that recovered when tissues were homogenized in low-ionic strength media (i.e., 0.32 M sucrose-20 mM Hepes). The data obtained represent the first quantitative estimates of immunoreactive Type II PLA 2 in human amnion and choriodecidua, and support the conclusion that previous analyses of the PLA 2 enzymatic activity present in gestational tissues have essentially excluded the contribution made by this PLA 2 isozyme to net enzymatic activity. We suggest that this isozyme represents a major component of the PLA 2 enzymatic activity present in human gestational tissues at term and that it contributes significantly to the phospholipid metabolism and arachidonic acid release which occurs during late pregnancy and at the time of labour.

Type II phospholipase A 2 in human gestational tissues: Subcellular distribution of placental immuno- and catalytic activity

The aims of this study were to determine the Subcellular distribution of Type II phospholipase A 2 immunoactivity (irPLA 2) and in vitro net PLA 2 catalytic activity in human term placenta and to establish the efficacy of previously utilised homogenisation procedures with respect to the quantitative recovery of Type II PLA 2 immunoreactive and in vitro net PLA 2 catalytic activity. Type II PLA 2 immunoactivity and PLA 2 catalytic activity recovered in 900 × g supernates prepared from placental tissue ( n = 3) homogenised in low ionic strength media (sucrose 0.32 M Hepes 20 mM; phosphate-buffered saline or phosphate-buffered saline containing 3 mM EGTA) was less than 10% of that recovered following homogenisation in high ionic strength medium (ammonium sulphate 10%, w/v). The Subcellular distribution of Type II PLA 2 immunoactivity and PLA 2 catalytic activity was established by the differential centrifugation (10 000, 20 000 and 100 000 × g) of placental homogenates ( n = 3). Although Type II PLA 2 immunoactivity was equally distributed throughout the particulate Subcellular fractions examined, PLA 2 catalytic activity increased by comparison in 100 000 × g particulate material. This apparent dissociation between irType II PLA 2 and catalytic activity may indicate the presence of other types of PLA 2 in this fraction. The data obtained in this study indicate that previous studies which have utilised low ionic strength extractions of human gestational tissue to characterise PLA 2 catalytic activity and Subcellular distribution have largely excluded the contribution made by Type II PLA 2. Consequently, much of the available published data on the role of PLA 2 in human parturition is inadequate. A reappraisal of this enzyme's contribution to the biochemical events associated with human pregnancy and labour is required.

Guanidinium derivatives act as high affinity antagonists of Na+ ions in occlusion sites of Na+,K(+)-ATPase.

We have screened various alkyl- and arylguanidinium derivatives as possible competitors of Na+ or Rb+ for the cation sites on renal Na+,K(+)-ATPase. Alkyl-monoguanidinium or alkylbisguanidinium (BisG) compounds (chain lengths of C3 to C10) competitively inhibit the occlusion of Rb+ and Na+ with an order of affinities C10 greater than C8 greater than C6 greater than C4 greater than C3. BisG compounds are approximately twice as effective as the equivalent alkylmonoguanidinium compounds. In media of high ionic strength, affinities of tens of micromolar are observed, e.g. 26 microM for BisG 8. m-(mXBG)- and p-xylylenebisguanidinium were synthesized and were found to compete with Rb+ or Na+ with intrinsic affinities of 7.7 and 8.2 microM, respectively. The hydrophobicity rather than the degree of proximity of the guanidinium groups in all BisG compounds appears to determine the binding affinity. A systematic search has been made of conditions in occlusion assays for which the inhibitor affinities are highest. When the pH is raised from 7.0 to 8.5, a 5-fold increase in affinity is observed, suggesting that the guanidinium derivatives compete with protons at sites of pKa approximately 7.5. Replacing Tris-HCl with choline chloride-containing media raised apparent affinities approximately 2-fold. All guanidinium derivatives stabilize the E1 conformation of fluorescein-labeled Na+,K(+)-ATPase, acting as competitive Na+ analogues. In media containing only 1 mM Tris-HCl, pH 8.55, very high affinities were observed for binding to the fluorescein-labeled enzyme (e.g. 0.08 microM for mXBG). In very low ionic strength medium, the inhibition was still competitive with Rb+ ions. However, there was also evidence for nonspecific adsorption to the membranes. The following findings show that mXBG, a typical guanidinium derivative, behaves as a Na(+)-like antagonist. (a) It inhibits Na+,K(+)-ATPase activity, competing strongly with Na+ but only weakly with K+ ions. (b) It inhibits phosphorylation from ATP, competing with Na+ ions. (c) Like Na+ ions, it blocks phosphorylation from inorganic phosphate. Based on these results, we propose that the guanidinium group binds to a relatively wide vestibule at the cytoplasmic surface; but, unlike Na+ or K+ ions, it cannot pass into a narrower region of the cation transport path within the membrane. Therefore, it blocks the occlusion and active transport of cations. In the future, high affinity guanidinium derivatives may serve the purpose of locating cation-binding domains of the pump protein after being converted to reactive affinity or photoaffinity covalent labels.

Advances in chelating exchange ion chromatography for the determination of trace metals using dye-coated columns

An ion chromatography system using chelating dye-coated columns for the determination of trace metals in high ionic strength media has been developed. A post-column spectrophotometric detection systems was employed to investigate transition and alkaline earth metals. Initial investigations involved a study of the effect of eluent pH, with various metal ions, on different dyes and substrates. Later work concentrated on separation of metal ions using dye-coated high-performance substrates, and a comparison was made with a commercial chelating column. A number of metal ion separations in 1 mol dm–3 potassium nitrate have been achieved under isocratic and gradient conditions.

Cortical granule matrix disassembly during exocytosis in sea urchin eggs

Cortical granule exocytosis in sea urchins was studied using hyperosmotic and polymer-containing seawater to halt granule matrix dispersal. Addition of Na 2SO 4-containing seawater (2.5 osmole/kg) to Strongylocentrotus purpuratus eggs 10 to 40 sec after insemination resulted in arrest of the exocytic wave during propagation. EM examination of these eggs revealed that matrix disassembly occurred in distinct stages. In the earliest stage, granule-plasma membrane fusion had occurred, but the matrix remained completely intact. This early stage was observed in hyperosmotic media, either ionic or nonionic, suggesting that matrix hydration is required for disassembly and exocytic pore widening, but not for membrane fusion. Subsequent stages, in which partially disassembled matrices remained within ω-configured pockets, were captured by activating eggs in 30% dextran in seawater. Stability of these intermediates stages required the presence of Ca 2+ and Mg 2+; in the absence of divalent cations the matrices completely disassembled and the exocytic pockets flattened. Divalent cations appeared to prevent fragmentation of the matrix lamellae. Late stages of matrix disassembly, in which the lamellae fragmented and formed small particles, were inhibited by media of high ionic strength. Hyperosmolality alone, provided by sucrose, was unable to halt these late stages suggesting that water availability does not play an important role once a critical point in matrix dispersal has been reached.

Treatment of sperm with high-ionic strength medium increases microsurgical fertilization rates of rabbit oocytes fertilized by subzonal placement of sperm

This study was conducted to investigate the requirement for sperm processing in microsurgical subzonal placement of sperm in rabbit oocytes. Fertilization rates with standard in vitro fertilization and microsurgical subzonal sperm placement were found to be similar (56 and 55%) when sperm treated with high-ionic strength Brackett's defined medium to initiate capacitation were used. Statistically significant reductions in fertilization rates for both standard in vitro fertilization and subzonal placement were noted when twice-washed spermatozoa were used. Initiation of capacitation of spermatozoa results in higher fertilization results even when the zona pellucida is bypassed during fertilization.

Stability of the acrosome of the brush-tailed possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) in vitro and after exposure to conditions and agents known to cause capacitation or acrosome reaction of eutherian spermatozoa

Ejaculated spermatozoa from brush-tailed possums and tammar wallabies were washed by a 'swim up' procedure into Hanks Balanced Salt Solution (HBSS), and then exposed to test solutions. Spermatozoa were incubated at 33 degrees C, or room temperature when long-term sperm survival (greater than 10 h) was required. Exposure of spermatozoa to calcium ionophore A23187, cyclic nucleotides, phosphoinositide pathway intermediates, lysophospholipids, trypsin or 'capacitating' high ionic-strength medium (380 mosmol) followed by 3% bovine serum albumin for periods up to 24 h did not induce acrosomal loss. However, there were major changes within the acrosome: large numbers of empty membrane-bound vesicles were formed, the electron density of the acrosomal matrix decreased and the acrosome swelled slightly. The origin of the vesicles is unclear but the acrosomal membranes and the plasma membrane remained intact.

The nonactivated progesterone receptor is a nuclear heterooligomer.

The discovery of the nuclear localization of estradiol and progesterone receptors in the absence of the steroid hormone has led to reconsideration of the model of cytoplasmic to nuclear translocation of these receptors upon exposure to hormone. Unoccupied nonactivated receptors are thought to be weakly bound to nuclei of target cells from which they are leaking during tissue fractionation and thus found in the cytosol fraction of homogenates in a nontransformed heterooligomeric "8-9 S" form, which includes hsp90. However, no direct biochemical evidence has yet been obtained for the presence of such heterooligomers in the target cell nucleus, possibly because it dissociates in high ionic strength medium used for extraction of the nuclear receptor. We took advantage of the combined stabilizing effects of tungstate ions and antiprogestin RU486 to extract a nuclear non-DNA binding nontransformed 8.5 S-RU486-progesterone receptor complex from estradiol-treated immature rabbit uterine explants incubated with the antagonist. As demonstrated by immunological criteria and by irreversible cross-linking with dimethylpimelimidate, the complex contained, in addition to the hormone binding unit, hsp90, and p59, another nonhormone binding protein. Control experiments carried out with the progestin R5020 yielded the expected nuclear transformed DNA binding 4.5 S-R5020-progesterone receptor complex. These results offer evidence for two distinct forms of steroid receptor in target cell nuclei. Besides the classical "4 S" agonist-receptor complex, tightly bound to the DNA-chromatin structure and in all probability able to trigger the hormonal response, we have observed in the RU486-bound state a non-DNA binding nontransformed 8.5 S form, presumably already present in the nucleus in the absence of hormone and representing the native nonactive form of the receptor.

Preparation of mitochondrial DNAs from sunflowers (Helianthus annuus L.) and from beets (Beta vulgaris) using a medium with a high ionic strength

The isolation of mitochondria from sugar beet and sunflower has been carried outina buffered medium of high ionic strength (I=1.40 M). Them tDNAs obtained were enriched for supercoiled molecules of low molecular weight. Chloroplast and mitochondrial DNA were successively prepared and used from a single leaf preparation for further analysis of restriction fragment length polymorphism. The few available wild relatives of sugar beet or sunflower have also been analyzed by comparison of the restriction patterns of their chloroplast and mitochondrial DNAs.

Free‐flow electrophoresis under microgravity: Evidence for enhanced resolution of cell separation

A mixture of fixed rabbit, guinea pig and rat erythrocytes, suspended in a relatively conductive solution, was separated by means of continuous free flow electrophoresis (CFFE) under 1 g- and microgram- conditions using a specially designed electrophoretic module. Short duration microgram conditions were realized on board a sounding rocket. Due to the energy input and the associated thermal convection a separation of the three differently charged cell types in distinct peaks was not possible under 1 g-conditions as shown by reference experiments on the ground before launch. In contrast to the poor resolution under 1 g-conditions, clear separation of the cell mixture could be recorded after lift-off of the rocket under microgram-conditions. Repeated measurements demonstrated that the separation profile was completely stable during the entire microgram-phase of about 6 min. Since the CFFE experiment in space was an exact replica of the ground reference experiments, the results demonstrated unambiguously the potential of CFFE for cell separation under microgram-conditions in media of high ionic strength.