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PubMed1966

Purification and characterization of phthalanilide--lipid complexes from tissues.

What appears to be a new class of phospholipids has been isolated from dog brain in the form of complexes with a substituted phthalanilide. The complexes were extracted by chloroform-methanol and purified by countercurrent distribution in solvent systems containing water, chloroform, methanol, and Freon 113. The binding of the phthalanilide congener to lipids has some ionic character. Cations such as H(+) or Ca(++) displaced the phthalanilide from its lipid complex. The pH for 50% displacement acid was about 3.8 and was independent of the purity of the complex. Thin-layer chromatography of the lipid yielded four subfractions of lipid, three of which were ninhydrin-positive and all of which yielded a group of unidentified ninhydrin-positive components on hydrolysis. Each lipid subfraction contained nitrogen, phosphorus, fatty acids, and glycerol but in different ratios. Of the known phospholipids containing nitrogen, none matches the composition and behavior of the lipids isolated as phthalanilide complexes. We have therefore concluded that the phthalanilides bind to a new class of phospholipids characterized by a high content of unidentified ninhydrin-positive components.

Anilides↗

PubMed1979

Fractionation of membranes from Acholeplasma laidlawii A on the basis of their surface properties by partition in two-polymer aqueous phase systems.

Acholeplasma laidlawii A consists of pleomorphic cell clusters surrounded by a single membrane. When lysed, a cell gives rise to several membrane fragments which cannot be separated from each other by isopycnic sucrose gradient centrifugation. A heterogeneous lateral organization of the cell membranes was detected by countercurrent distribution of membrane fragments in a two-polymer aqueous phase system. It revealed that the membranes consist of at least two subpopulations with respect to surface properties. Changes in the fatty acid and cholesterol content of the membranes revealed that the resolution of different subpopulations was predominantly due to a critical ratio of monoglucosyldiglyceride to diglucosyldiglyceride. The heterogeneity of the membrane probably depends on lipid-lipid and lipid-protein steric interactions. Charged lipids, an apolar monoglucolipid and the ratio between lipids and proteins also affect membrane partition. The differences in the subpopulations were further reflected by different specific activities of NADH dehydrogenase, NADH oxidase and ATPase. These activities varied independently. Minor quantitative differences in the protein patterns of different subpopulations were apparent. The origin and the preservation of the membrane subpopulations are discussed in terms of lipid-lipid and lipid-protein interactions, their age and energy metabolism.

Acholeplasma laidlawii↗

PubMed1977

[Primary structure of tRNA Thr 1a and b from brewer's yeast].

One of the two major species of brewer's yeast tRNA threonine (tRNA Thr 1) has been purified by countercurrent distribution followed by two chromatographic steps (respectively on a Sepharose 4B and a BD-cellulose column). Complete digestion with pancreatic and T1 RNases and a partial hydrolysis with T1 RNase followed by the isolation and determination of the nucleotide sequences of the resulting fragments permitted the derivation of its primary structure. tRNA Thr 1 is in fact a mixture of two subspecies differing only by a A49-U65 base pair in 50 per cent of the molecules which is replaced by a G49-C65 pair in the other 50 per cent. These two subspecies consist of 76 nucleotide residues including 14 minor nucleotides. They show a characteristic m3C at the 3'terminal end of the anticodon loop, an anticodon I-G-U followed by t6A and C48, uncompletely modified (50 per cent) to m5C within the 5 nucleotides long extra-arm. The minor nucleotides m2G m2 2G are located at positions in which they generally occur in the tRNA structures as does m1A within the T-psi-C loop.

Base Sequence↗

PubMed1983

Surface properties of lymphocyte subpopulations in autoimmune NZB/NZW F1 hybrid mice: alterations correlated with the immunodeficiency of aging.

Partitioning in a two-polymer aqueous phase system was used to probe the surface properties of lymphoid cell subpopulations in aged male NZB/NZW F1 hybrid (B/W) mice, an important model of autoimmunity, immunodeficiency, and lymphoid malignancy. Spleen cells were fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged dextran-polyethylene glycol system. CCD of spleen cells from young, clinically normal male B/W mice yielded several broad distribution patterns which frequently had two or more peaks. Analysis of differentiation antigens and functional properties of cells from different parts of the distribution revealed a subfractionation of the three major lymphocyte subpopulations. B lymphocytes had a low partition coefficient (K); T cells had an intermediate K and null cells had the highest K. To examine the partitioning behavior of T lymphocytes, spleen cells which were nonadherent to nylon wool columns were subjected to CCD. Nonadherent cells from young B/W mice consistently gave a single peak with high K. Aged mice (18 months) usually had nonadherent cells with a predominantly low K. In some experiments a systematic increase in the number of these cells could be demonstrated with increasing mouse age. An analysis of the adherence and partitioning behavior of lymphocyte subpopulations revealed no change in the adherence properties or proportions of B lymphocytes in aged mice. The large proportion of cells having a low partition coefficient in the nonadherent spleen cell population of old mice appears to be due to an increase in the number of null cells and in a decrease in the K of some T lymphocytes.

Aging↗

PubMed1976

Properties of chloroplasts isolated by phase partition.

Chloroplasts from spinach can be separated into at least three different populations by countercurrent distribution using polymer two-phase systems. The chloroplast particles of the three populations differ in protein/chlorophyll ratio, ultrastructure and metabolism. One population, peak I, consists of intact chloroplasts surrounded by the chloroplast envelope; the second population, peak II, consists of chloroplasts, which have lost their envelopes and much of their stromal material; the third population, peak III, consists of particles containing intact chloroplasts surrounded by a membrane-bound cytoplasmic layer including mitochondria and peroxisomes. Rapid batch procedures of peak I chloroplasts incorporated 14C almost entirely into glycolate and intermediates of the Calvin cycle and starch synthesis. Only small amounts were found in sucrose and amino acids. On the other hand preparations of peak III chloroplasts have a much broader spectrum of 14C-labelled products. Sucrose, malate and some amino acids contained about 40% of the 14C incorporated. It is concluded from these experiments that sucrose is formed not within the chloroplast but in the cytoplasm from intermediates exported by the chloroplast. The origin of peak III particles and their use for studying the cooperation between the chloroplast and the surrounding cytoplasm including mitochondria and peroxisomes is discussed.

Carbon Dioxide↗

PubMed1997

Two populations of the estrogen receptor separated and characterized using aqueous two-phase partitioning.

Two populations of the rat uterine estrogen receptor (ER) were separated and characterized using aqueous two-phase partitioning. Countercurrent distribution of rat uterine cytosolic ER allowed rapid and efficient separation of two populations, one population partitioned preferentially into the top phase (T, K(obs) = 3-6) and the other into the bottom phase (B, K(obs) = 0.01-0.03). The majority of unoccupied cytosolic ER is in the T population. Upon estrogen binding and/or heating to 30 degrees C in vitro the T population is converted to the B population. The transition from T to B does not exclusively involve loss of heat shock protein 90 and does not alter the ligand binding ability of the steroid binding domain. Using the human ER steroid binding domain overproduced in Escherichia coli and the steroid binding domain generated by partial trypsinization of the rat uterine ER, we demonstrate that the characteristic distinguishing T and B populations is not localized to this domain alone but may be associated with the amino terminal half of the ER (the A/B and DNA binding domains). The T to B transition of the ER also occurs in human MCF-7 breast cancer cells upon treatment with estrogen at 37 degrees C.

Animals↗

PubMed1983

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. I. Human red blood cell subpopulations.

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained. It was further determined, by repartitioning 51Cr-labeled cells from the left or right ends of a CCD of human red blood cells admixed to unlabeled unfractionated erythrocytes, that a subpopulation with higher partition coefficient exists (probably constituting the old red cells). These experiments serve to illustrate (a) that human red blood cells, contrary to a previous report, can be subfractionated by partitioning and (b) the usefulness of this new method in detecting smaller surface differences between closely related cell populations than was heretofore possible by partitioning alone.

Cell Separation↗

PubMed1969

Studies on phenolic steroids in human subjects. IX. Role of the intestine in the conjugation of estriol.

In order to compare the enteric circulation of estriol-16alpha-glucosiduronate (see preceding paper) with that of estriol (E(3)), labeled estriol was administered to six women by several routes: both injection and infusion (300 min) into the cubital vein, injection into the portal vein system, ingestion and instillation into the jejunum and ileum. Urine, collected from 0-2, 2-4, 4-8, 8-12, and 12-24 hr, was analyzed by countercurrent distribution for its content of radioactive 3- and 16-glucosiduronate (E(3)-3Gl,E(3)-16Gl) and sulfoglucosiduronate (E(3)-3S,16Gl) of estriol. After peripheral injection of E(3), E(3)-16Gl was excreted rapidly and E(3)-3S,16Gl at a slower and more constant rate. E(3)-3Gl was barely detectable after infusion. After injection of E(3) into the portal vein, the excretion of E(3)-3S,16Gl was greater and quicker than after peripheral injection. Even in a subject with a complete bile fistula, the urinary excretion of E(3)-3S,16Gl was essentially unchanged. Ingestion also produced the same result. Only after instillation into the ileum was a large and rapid excretion of E(3)-3Gl obtained, whereas the excretion of E(3)-3S,16Gl, and E(3)-16Gl were depressed. These results together with those of the preceding paper suggest that E(3) does not readily appear in the small intestine except via a hepatoenteric circulation that produces very little E(3)-3Gl. When present in the distal segment of the small intestine, however, absorption, conjugation, and elimination proceed readily.

Biliary Fistula↗

PubMed2003

Different functional states of ram spermatozoa analysed by partition in an aqueous two-phase system.

The surface of spermatozoa plays a critical role in many stages involved in fertilisation. The plasma membrane undergoes important alterations in the male and female reproductive tract, which result in the ability of spermatozoa to fertilise eggs. One of these membrane modifications is sperm capacitation, a process by which sperm interacts with the zona pellucida receptors leading to the acrosome reaction. It has been proposed that the freezing process induces capacitation-like changes to spermatozoa, and that this premature capacitation could explain the reduction in longevity and fertilising capacity of cryopreserved mammalian spermatozoa. Our research focused on the relationship between membrane alterations occurring throughout freezing-thawing and the processes of capacitation and acrosome reaction. We used centrifugal countercurrent distribution (CCCD) analysis to compare the partition behaviour of ram spermatozoa that was either subjected to cold-shock or frozen-thawed with capacitated and acrosome reacted samples. In addition, the effect of the induced acrosome reaction on membrane integrity of ram spermatozoa was studied using biochemical markers and electron microscopy scanning. The CCCD analysis revealed important similarities between the surface characteristics of capacitated and cold-shocked sperm as well as between acrosome-reacted and frozen-thawed sperm. Cold-shocked and capacitated sperm showed an increased cell affinity for the lower dextran-rich phase as well as a decreased heterogeneity. Likewise, the induction of the acrosome reaction resulted in a loss of viability and an important decrease in cell surface heterogeneity compared to the untreated-control sample. Similar surface changes were found when semen samples were frozen with either Fiser or milk-yolk extender. These results confirm those obtained for membrane integrity by fluorescence markers. Thus, the high cell viability value found in the control sample (74.5%) was greatly decreased after cold-shock (22.2%), cryopreservation (26.38% Fiser medium, 24.8% milk-yolk medium) and acrosome reaction (6.6%), although it was preserved after inducing capacitation (46.7%). The study using electron microscopy scanning revealed dramatic structural alterations provoked by the induction of the acrosome reaction.

Acrosome Reaction↗

PubMed1975

Abnormal membrane surface properties during maturation of rat reticulocytes elicited by bleeding as measured by partition in two-polymer aqueous phases.

Partition of cells in two-polymer aqueous phases is an extremely sensitive method for the separation of cells and for tracing subtle changes in the cells' membrane surface properties (primarily surface charge) as a function of in vivo processes (e.g. differentiation, maturation, ageing). Combining isotope labelling and cell countercurrent distribution techniques we have examined the membrane surface properties of rat reticulocytes produced in response to erythroid stress by bleeding. It was found that the rapid increase in the partition coefficient of normal reticulocytes subsequent to release into the peripheral blood (which reflects a rapid increase in membrane surface charge) is absent in reticulocytes produced in response to bleeding. In this way the reticulocytes behave like those produced in response to repeated phenylhydrazine injections. 'Stress reticulocytes' never mature to erythrocytes having normal membrane surface properties as judged by partition. The experiments show that our previous results are not due to the phenylhydrazine per se but rather to the production of 'stress reticulocytes'. Further, if remodelling of the phlebotomy-induced reticulocyte cell membrane occurs in the circulation as has been suggested it does not lead to a cell with normal membrane surface properties. Whether the abnormal membrane surface properties of 'stress reticulocytes' and of the erythrocytes to which they give rise affect the cells' life-span is not clear since phenylhydrazine-induced 'stress reticulocytes' have been reported to have at least a component of short-lived cells while the phlebotomy-induced cells are said to have a virtually normal life-span. It is likely that reticulocytes produced in severe anaemias, in general, mature to erythrocytes having abnormal surface properties (i.e. lower membrane surface charge).

Animals↗

PubMed1969

Studies on phenolic steroids in human subjects. 8. Metabolism of estriol-16 alpha-glucosiduronate.

6,7-(3)H-Estriol-16alpha-glucosiduronate-(14)C was administered to eight women (nine studies) by several routes: both injection and infusion (300 min) into the cubital vein, injection into the portal vein system, ingestion and instillation into the duodenum, jejunum, and ileum. Urine, collected from 0-2, 2-4, 4-8, 8-12, and 12-24 hr, was analyzed by countercurrent distribution for its content of radioactive 3- and 16-glucosiduronate (E(3)-3Gl,E(3)-16Gl) and sulfoglucosiduronate (E(3)-3S,16Gl) of estriol as well as for (3)H/(14)C ratio of each conjugate. After peripheral injection 50-60% of the injected E(3)-16Gl was excreted unchanged along with about 5% as E(3)-3S,16Gl with an unchanged (3)H/(14)C ratio, indicating direct sulfation of the injected E(3)-16Gl. During a 300 min infusion, urinary excretion closely resembled that following injection. But 2-4 hr after the end of the infusion excretion of E(3)-3S, 16Gl stopped, excretion of E(3)-3Gl (17%/24 hr) with an elevated (3)H/(14)C ratio started, and excretion of E(3)-16Gl continued (70%/24 hr), but with a rapidly increasing (3)H/(14)C ratio. This indicated sequestration in a sluggishly metabolizing compartment where two processes occurred: (a) extensive hydrolysis of E(3)-16Gl followed by reconjugation at either C3 or C16 with unlabeled uridine diphosphate glucuronic acid (UDPGA), thereby increasing the (3)H/(14)C ratio; and (b) transconjugation from C16 to C3, thereby producing E(3)-3Gl with finite (3)H/(14)C ratios. Instillation into various segments of the small intestine produced results qualitatively similar to those after intravenous infusion, whereas ingestion and intraportal injection resembled peripheral intravenous injection. Therefore, we have postulated the possibility of an enteric circulation (in addition to an enterohepatic circulation) in which the steroid or its conjugates are transported into the small intestine in the succus entericus, modified, and then reabsorbed and excreted in the urine-a process which requires several hours.

Adolescent↗

PubMed1975

Affinity partitioning. A method for purification of proteins using specific polymer-ligands in aqueous polymer two-phase systems.

We describe a method, called affinity partitioning, for the purification of proteins containing specific ligand binding receptor sites. This method adds specificity to the procedures for protein purification with aqueous polymer two-phase systems by introduction of a polymer derivative, coupled to an appropriate ligand. The addition of a polymer-ligand that partitions predominantly into one phase shifts the protein that binds this substance to the same phase. By performing countercurrent distribution in the presence of a polymer-ligand, the protein that binds the polymer-ligand can be separated from a heterogenous mixture. One example of affinity paritioning used dextran as the polymer-ligand. Dextran was chosen since it is a constituent of the most commonly used system for partitioning proteins. In a dextran-poly(ethylene oxide) system, concanavalin A bound dextran and partitioned predominantly into the dextran-rich phase. The addition of the specific competitor, D-mannose, displaced the partition coefficient toward unity, while the application of L-fucose, a noncompetitor, had little effect. Application of affinity partitioning to the purification of another protein required the synthesis of a specific polymer-ligand. To study this we synthesized dinitrophenyl-poly-(ethylene oxide), which binds specifically to S-23 myeloma protein. Addition of dinitrophenyl-poly(ethylene oxide) to the dextran-poly(ethylene oxide) phase system shifted the S-23 myeloma protein into the poly(ethylene oxide)-rich phase. epsilon-N-dinitrophenyl-L-lysine, by competing with binding of dinitrophenyl-poly(ethylene oxide), antagonized the latter's effect on the partition coefficient of S-23 myeloma protein. By adding various amounts of dinitrophenyl-poly-(ethylene oxide), we correlated the partition coefficient with concentration of polymer-ligand. A model of the action of polymer-ligand derivatives on the partition coefficient, derived from thermodynamic considerations, was found to be consistent with the experimental data relating the concentration of polymer-ligand and partition coefficient. Affinity partitioning should prove to be a useful complement to affinity chromatography in the purification of mixtures of proteins. Since cells and subcellular particles may be purified with aqueous polymer two-phase systems, affinity partitioning might be applied to their fractionation by using polymer-ligands specific for unique surface receptors.

Animals↗

PubMed1992

Effect of cell exposure to top or bottom phase prior to cell partitioning in dextran-poly(ethylene glycol) aqueous phase systems: erythrocytes as a model.

Cells exposed to dextran (Dx)-rich bottom phase prior to cell partitioning in Dx-poly(ethylene glycol) (PEG) aqueous two-phase systems have lower partition ratios than cells exposed to PEG-rich top phase. Aspects of this previously observed phenomenon were explored. In the present work charge-sensitive phases made with Dx T500 and PEG 8000 were used exclusively. It was found that: (1) even on countercurrent distribution (CCD) red cells (RBC) loaded in bottom phase have a lower apparent partition ratio, G, than the same cells loaded in top phase; (2) when part of the same cell population is loaded into top phase and part into bottom phase of the same load cavities for CCD, with the cells loaded into top or bottom bearing an isotopic tracer (51Cr), the cells loaded into top phase have a higher G value than the cells loaded into bottom phase; (3) the shift in the CCD curves of human or of rat RBC between cells loaded in top or bottom phase using systems having the same polymer concentration (though different salt compositions) shows no striking difference and is, for the number of experiments run, not statistically significant; (4) when the quantity of cells loaded for CCD is reduced from 10(9) to 10(8), the G value of cells loaded in top phase is reduced slightly while that of cells loaded in bottom phase is diminished more appreciably; (5) increasing polymer concentrations yield larger differences in G values between (rat) RBC loaded in top or bottom phase; (6) when cells exposed to top or bottom phase, respectively, are centrifuged and suspended in bottom or top phase, respectively, their CCD patterns are qualitatively similar to cells exposed to these latter respective phases initially; (7) rat RBC populations containing 59Fe-labeled cells of different but distinct age are fractionated on CCD irrespective of whether loaded in top or bottom phase. An exception are populations containing very young mature labeled cells (e.g., 4-d old) which are resolved when loaded in top phase but not in bottom phase. Thus cell populations exist which can be resolved by CCD when loaded in one of the phases but not when loaded in the other. Glutaraldehyde-fixed rat RBC containing 4-d old labeled cells are fractionated by CCD irrespective of whether loaded in top or bottom phase.

Animals↗

PubMed1992

Aqueous two-phase systems for biomolecule separation.

Over the past thirty years, aqueous polymer two-phase technology has evolved, both experimentally and theoretically, into a separation science with many useful applications in biomolecule purification and bioconversion. This paper summarizes the developments in the applications of aqueous two-phase systems to biotechnology. The main topics to be considered are the phase diagram and its characteristics, fundamentals of biomolecule partition, large-scale and multi-stage aqueous two-phase biomolecule purification, and extractive bioconversions. The first topic involves a discussion of the thermodynamics of aqueous polymer two-phase formation and how it is influenced by such factors as polymer molecular weight and concentration, temperature, and salt type and concentration. Next, the theoretical and experimental aspects of biomolecule partition in aqueous two-phase systems will be discussed in light of the factors which influence biomolecule partition: polymer concentration and molecular weight; temperature; salt type and concentration; the addition of charged, hydrophobic and affinity derivatives. Having reviewed the fundamentals of phase diagram formation and biomolecule partition, the next two topics are applications of aqueous two-phase technology. The first set of applications involve the large-scale extraction of proteins using one to three equilibrium stages and multi-stage purifications using countercurrent distribution, liquid-liquid partition chromatography and continuous countercurrent chromatography. The second application, and very promising area for future aqueous two-phase technology, is the extractive bioconversion which permits the simultaneous production and purification of a biomolecule.

Biotechnology↗

PubMed1982

Microcirculation of left atrial muscle, cerebral cortex and mesentery of the cat. A comparative analysis.

By means of transillumination (microtransilluminator and light pipe), comparative analyses were carried out on geometry, topography, and morphometry of microcirculation in the cerebral cortex, left atrial muscle, and mesentery of the cat using computer analysis. In addition, specific types of capillary distribution (concurrent, countercurrent, and asymmetric distribution) in these three organs were ascertained from images visualized on films. These parameters were related to their role in tissue oxygen supply. It was found that mean capillary diameter, mean intercapillary distance, total capillary length, and total capillary surface area differed significantly among the three organs. Differences in mean capillary tortuosity between cerebral and left atrial muscle and between left atrial muscle and mesentery also were significant. Mean capillary tortuosity in mesentery and cerebral cortex was of equal magnitude. In the cerebral cortex, a high degree of tortuosity and asymmetric capillary distribution favor tissue oxygenation. A similar situation exists in left atrial muscle, although some concurrent and countercurrent distribution could be detected. In the mesentery, the combination of high capillary tortuosity and concurrent capillary arrangement is unfavorable for tissue oxygenation.

Animals↗

PubMed2003

i-SNAREs: inhibitory SNAREs that fine-tune the specificity of membrane fusion.

A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.

Binding Sites↗

PubMed2000

Multistage electrophoresis system for the separation of cells, particles and solutes.

It is common to operate equilibrium-based separation methods, such as distillation and extraction, as multistage unit operations, in which equilibrium is presumably achieved within each stage. Two rate-based separation processes, free electrophoresis and magnetic particle separation, have now been operated in multistage mode. Preparative free electrophoresis of particles and solutes has resisted scale-up and is confined to a narrow range of ionic compositions. Natural convection induced in electrophoresis buffers by Ohmic heating has been a strong deterrent and has led to such measures as radial electrophoresis in Couette flow, free-flow electrophoresis, low-gravity electrophoresis, density gradient electrophoresis, and reorienting density gradient electrophoresis, to name a few. The short vertical electrophoresis path exploited in the last-mentioned forms the basis for multistage electrophoresis. A thin-layer countercurrent distribution apparatus was designed and constructed so that up to 20 fractions could be collected on the basis of electrophoretic mobility by applying an electric field. The mixture to be separated starts in a bottom cavity, and successive top cavities collect fractions as separand particles or molecules are electrophoresed upward out of the bottom cavity. Mathematical models of this process were developed, and experiments were performed to verify the predictions of the models by collecting and counting particles in each cavity after fractionation.

Animals↗

PubMed1990

Small intestinal differentiation in human colon carcinoma HT29 cells has distinct effects on the lateral diffusion of lipids (ganglioside GM1) and proteins (HLA class 1, HLA class 2, and neoplastic epithelial antigens) in the apical cell membrane.

We have studied the effect of maturation to small intestinal-like epithelial cells of the human colonic carcinoma cell line HT29 on the lateral mobility of different representative membrane components (lipid, proteins), as assessed with fluorescence recovery after photobleaching (FRAP). Maturation was induced in vitro in the HT29 cells by replacing glucose (Glu) with galactose (Gal) in the growth medium (DMEM) during a 21-day period. Scanning electron microscopy revealed an increased number of microvilli in the apical cell membrane, and enzyme analyses (alkaline phosphatase, aminopeptidase) in combination with aqueous countercurrent distribution, indicated that maturation was induced with DMEM-Gal. In comparison to control cells grown in DMEM-Glu medium, the more small intestinal-like cells grown in DMEM-Gal displayed no alteration of the lateral mobility of either cholera toxin (B subunit)-labelled ganglioside GM1 (diffusion coefficient, D [x 10(8)] = 0.8-0.9 cm2s-1; mobile fraction, R = 50-60%) or antibody-stained Class 2 histocompatibility (HLA-DR) antigen (D [x 10(9)] = 2 cm2s-1; R = 60-70%). However, antibody-labelled beta 2-microglobulin of HLA Class 1 antigen displayed increased mobility in HT29-Gal cells; D was x 1.4 and R x 1.8 larger in the HT29-Gal cells. By contrast, the mobility of a neoplastic antigen was reduced; D and R were x0.60 and x0.69 of the values seen in HT29-Glu cells. It is thus concluded that DMEM-Gal-induced differentiation in confluent HT29 cells is accompanied by specific rather than general effects on the lateral mobility of different membrane components.

Carcinoma↗