Partition of ribosomes in two-polymer aqueous phase systems

Two-polymer aqueous phase systems are described in which ribosomes selectively partition into one of the phases. One of the phase systems is used to determine rapidly and conveniently binding of thiostrepton and erythromycin to Escherichia coli ribosomes under equilibrium conditions.     

Cell-cell affinity probed by countercurrent distribution in two-polymer aqueous phase systems

Aqueous solutions of dextran and of polyethylene glycol when mixed form immiscible liquid two-phase systems with a polyethylene glycol-rich top and a dextran-rich bottom. Such phases can be buffered and rendered isotonic and are suitable for the partition of cells. The partition coefficient of cells (i.e., their relative affinity for the top or bottom phase or their adsorption at the interface) depends on the polymer concentrations, on the ionic composition and concentration and, most sensitively, on their membrane surface properties. When two cell populations are mixed the partition coefficient of each is unaffected by the presence of the other population unless an interaction takes place between them. By countercurrent distribution of two cell populations, in a phase system selected such that the partition coefficient of one population is high (i.e., more cells in the top phase) and of the other low, one should be able to detect subtle interactions between cells in the two populations should they occur. Results of experiments with a model system consisting of human peripheral blood mononuclear cells and sheep red blood cells bear out the feasibility of this approach. At low ratios of sheep erythrocytes to mononuclear cells no interaction is apparent. With increasing ratios there is an increasing shift in the distribution curve of subpopulations of mononuclear cells (i.e., T-lymphocytes) as they interact with the sheep red cells. Substitution of rabbit red cells for sheep erythrocytes (at high ratios) reveals a small yet significant shift of a subpopulation of mononuclear cells as well. Distribution of human peripheral blood lymphocytes from patients with chronic lymphocytic leukemia (all B-lymphocytes) are essentially unaffected by the presence of sheep red cells. This sensitive new method, still in its infancy, holds out the hope for the detection of previously unknown cell-cell affinities and for probing suspected cell-cell interactions.     

Membrane surface properties of normal and malignant cells: partition in aqueous two-polymer phase systems

The partition of normal and malignantly transformed fibroblast lines and cell lines initiated from malignant human astrocytomas and a benign ganglioneuroma has been examined in aqueous dextran-polyethylene glycol phase system containing phosphate buffer with a low phosphate/sodium chloride ratio. The malignant astrocytomas showed a significantly lower partition coefficient as compared with the benign ganglioneuroma. Treatment of astrocytoma cells with dexamethasone caused an increase in the partitioning of the cell population. No differences were found in the partition behaviour of normal BHK-21 cells and their malignant transformants, the TRES fibrosarcoma cells. Polyoma and simian virus-transformed 3T3 fibroblasts showed partition ratios similar to the untransformed cells. Dexamethasone pre-treatment had no effect on the partition behaviour of these cells. The significance of these observations has been discussed in relation to the surface hydrophobicity and the neoplastic state.     

On the relation between surface area and partitioning of particulates in two-polymer aqueous phase systems

Partitioning in dextran-poly(ethylene glycol) aqueous two-phase systems is an established method for the separation of biomaterials. Size and surface properties are generally regarded as parameters which contribute to the determination of the materials' partition coefficients, K. While molecular weight or surface area can be one of the determinants of the K value of biomaterials in the size range of macromolecules to very small particulates (e.g. some viruses), partitioning liposomes of identical surface properties and different but distinct sizes indicate that surface areas greater than about 0.2 μm² do not affect the K value obtained. Analysis of available partitioning data of much larger particulates (i.e. cells) reveals that surface properties per unit area outweigh surface area per se in determining the K value in non-charge-sensitive, charge-sensitive and biospecific affinity phase systems.     

The dynamics of phase partition. A study of parameters affecting rat liver organelle partitioning in aqueous two-polymer phase systems.

Separation of subcellular organelles by two-phase partition is thought to reflect differential partition of the organelles between the two phases or between one of the phases and the interface. Studies by Fisher and colleagues [Fisher & Walter (1984) Biochim. Biophys. Acta 801, 106-110] suggest that cell separation by phase partition is a dynamic process in which the partition changes with time. This is mainly due to association of the cells with sedimenting droplets of one phase in the bulk of the other. Rat liver organelle partition was studied to determine whether the same dynamic behaviour is observed. Partition was clearly time-dependent during 24 h at unit gravity, and was also affected by altering the volume ratio of the two phases and the duration of phase mixing. These results indicate that, as with cells, the partition of organelles between phases is a dynamic process, and is consistent with the demonstration that organelles adhere to the phase droplet surfaces. Optimization of the volume ratio between phases may lead to significant processing economies. Organelle sedimentation in the upper phase was significantly faster than in the isoosmotic sucrose. Theoretical modelling of apparent organelle sizes indicates that aggregation occurs in the poly(ethylene glycol)-rich upper phase. This phenomenon is likely to limit the use of this technique in organelle separations unless means can be found to decrease aggregation.     

Cell separations and subfractionations by countercurrent distribution in two-polymer aqueous phase systems depend on non-equilibrium conditions

Certain kinetic aspects of the partitioning behaviour of erythrocytes in dextran-poly(ethylene glycol) aqueous phase systems have been examined, and their implications for cell partitioning have been considered. It is concluded that the diverse and sometimes unique fractionations attained by use of multiple extractions (e.g., countercurrent distribution) of cells in two-polymer aqueous-phase systems depend on non-equilibrium conditions.     

Subfractionation of human peripheral blood lymphocytes on the basis of their surface properties by partitioning in two-polymer aqueous phase systems.

Partitioning of cells in dextran-poly(ethylene glycol) aqueous-aqueous two-phase systems is a sensitive method for separating cells and for obtaining information on their surface properties. Highly purified lymphocytes were obtained by velocity sedimentation of human peripheral blood mononuclear cells and fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged two-polymer aqueous phase system. The lymphocytes remained viable after separation (order of 90%) and the E-rosetting cells responded (after adding back monocytes) to mitogens (PHA, Con A, PWM). Not only was the total lymphocyte population found to be highly heterogeneous (as evidenced by a broad and skewed distribution curve), but we were able to show that cells that rosetted with E, or had complement or Fc receptors were composed of additional subpopulations as well. The bulk of complement-receptor-bearing cells had the lowest partition coefficient (K), E-rosetting cells an intermediate K, and Fc-receptor-containing cells the highest K. The largest lymphocytes were among the subpopulation having the highest K and neither responded to T cell mitogens nor rosetted with E. Our results thus demonstrate that human peripheral blood lymphocytes can be subfractionated by CCD. The fractions are differentially enriched with lymphocyte subpopulations having characteristic surface markers and functional abilities.     

Various topochemical arrangements of sialic acids on human erythrocytes as detected by partition in aqueous two-polymer phase systems

Human and rabbit red blood cells were subjected to partition in an aqueous, buffered Ficoll-Dextran two-phase system. The effect of neuraminidase treatment on the cell partition behaviour was examined and compared with the amount of sialic acids released from the cell surface and with the change in the electrophoretic mobility of the cells. The data obtained in the study indicate that there are two main groups of sialic acids differing in their topochemical arrangement on the human erythrocyte surface, and their relative hydrophobicity was evaluated. The results obtained in the case of rabbit red cells seem to indicate that sialic acids present on the cell surface are not the only major ionogenic surface components as is the case for human red cells. The data obtained support the assumption that the membrane surface charge is the determinant of cell partition only as a factor affecting the relative hydrophobicity of the cell surface.     

Relative hydrophobicity of surfaces of erythrocytes from different species as measured by partition in aqueous two-polymer phase systems

Erythrocytes from different species were subjected to partition in an aqueous, buffered Ficoll/Dextran two-phase system. The effects of different salt composition of the phase system on the distribution of erythrocytes was examined. Different ratios of sodium chloride to sodium phosphate buffer (pH 7.4) with the ionic strength varying from 0.176 to 0.288 M were used in the systems and similar relationship between the partition coefficients of the cells under study and the ionic strength were established. The relationships were treated according to a general equation previously established (Zaslavsky, B.Y., Miheeva, L.M., Metechkina, N.M., Pogorelov, V.M. and Rogozhin, S.V. (1978) FEBS Lett. 94, 77-80) and the results obtained were used to evaluate the relative hydrophobicity of the cells' surface.     

Morphology, developmental ultrastructure and ultracytochemistry of staminal hairs in Bulbine inflata (Asphodelaceae) in relation to function

Results of light and electron microscopy and preliminary ultracytochemical studies of the staminal hairs of Bulbine inflata at different stages of development are reported here. The staminal filaments are covered with yellow, unicellular, linear, erecto-patent hairs. These staminal hairs arise directly as single cell outgrowths from epidermal cells of the filament. The surface of each hair is patterned with helical wall thickenings in an anticlockwise direction. This wall is covered by a thick folded cuticle, and formed of a loosely fibrillar cellulose layer. The hair cell possesses a cytoplasm rich in organelles. Especially ribosomes are abundant. Plastids contain large starch grains and peripheral lipid droplets. The smooth endoplasmic reticulum cisternae (SER) encircle the plastids and mitochondria; it is extended in the cytoplasm along the hair length. These hairs have functions in flower pollination attracting pollinators visually, secreting specific substances, providing increased surface area, protecting the filaments and being involved in their movement and vibration.     

Subcellular fractionation of rat liver homogenates using two-polymer phase systems in a toroidal-coil centrifuge.

The principal organelles of rat liver homogenates were fractionated by two-phase partition chromatography using toroidal-coil centrifugation with a mixture of dextran T 500 and poly(ethylene glycol) 6000 in 0.26 M-sucrose containing 10 mM-sodium phosphate/phosphoric acid buffer, pH 7.4. The effects of varying the following parameters on organelle elution profiles, as reflected by their marker-enzyme activities, were studied: centrifuge speed; the composition and relative proportion of dextran-rich and poly(ethylene glycol)-rich phases in the eluent; flow rate; sample volume; homogenate concentration; helix diameter; tubing bore and the number of loops in the coil. Optimal resolution of the organelles was achieved with a toroidal coil of internal diameter 1.07 mm with a 4.55 mm helix diameter on a 0.42 m-diameter rotor running at 1000 rev./min. The eluent was prepared by combining, in a ratio of 93:7 (v/v), the poly(ethylene glycol)-rich upper phase and dextran-rich lower phase obtained from a phase mixture containing 3.3% (w/w) dextran and 5.4% (w/w) poly(ethylene glycol). The flow rate of the eluent was 14ml/h. Optimal conditions for separation of the organelles were evaluated. Resolution of plasma membrane and lysosomes was achieved. Separation of endoplasmic reticulum, which showed marked heterogeneity, from plasma membrane was also demonstrated. DNA and marker enzymes for peroxisomes, mitochondria and cytosol showed distinct elution profiles.     

Predator-induced changes in fluted giant clam (Tridacna squamosa) shell morphology

Predator-induced defences have been demonstrated in numerous marine molluscs, but never in giant clams (Bivalvia: Tridacnidae). Water-borne cues from the predatory crab Myomenippe hardwickii were tested for their ability to induce changes in the shell morphology, strength and growth of juvenile fluted giant clams, Tridacna squamosa. Specimens were maintained in effluent from tanks holding ‘fed crabs’, ‘starved crabs’ and ‘no crabs’. To ensure changes were due to predator cues only, food for the crabs was provided ex situ and measures were taken to minimise the prey signal. After 182 days, MANOVA identified differences in various shell parameters relating to shape and strength. CDA showed a clear change in overall morphology, with strong separation among the three treatments. Effluent from ‘fed crabs’ had a greater influence than effluent from ‘starved crabs’, possibly because the starved crabs were perceived as weaker, and thus less risk. Significantly more clams died in ‘fed crabs’ treatments (25%) and ‘starved crabs’ treatments (30%) compared to the ‘no crabs’ control (0%). This degree of mortality has not been observed in similar experiments elsewhere and represents a new challenge to interpret. We suggest that the cause is unlikely to be related to the plastic responses, but rather a result of some form of crab-associated contaminant.     

Alterations in membrane surface properties during cell differentiation as measured by partition in aqueous two-polymer phase systems: Rat intestinal epithelial cells

Membrane surface properties of rat intestinal epithelial cells (crypt base to villus tips) were studied by cell partition in a two-polymer aqueous phase system. A higher partition generally reflects higher cell surface charge (or charge-associated properties) which is not necessarily the same as the charge determined by cell electrophoresis since the latter reflects only the charge at the plane of shear while the former gauges it deeper into the membrane [10]. Cells were prepared by the method of Weiser [22] which sequentially yields cell fractions from villus tips to crypt base. The isolated cells were subjected to countercurrent distribution in a dextran-polyethylene glycol aqueous two-phase system. Countercurrent distribution on the first fractions obtained by Weiser's method have a peak to the left and a smaller peak to the right indicating a surface membrane heterogeneity of upper villus cells; last fractions have a peak only to the right. When all fractions are pooled before countercurrent distribution two well-separated peaks are obtained with the right peak sometimes showing additional heterogeneities. Experiments combining isotope labeling of cells with countercurrent distribution lead us to conclude that the membrane charge (or charge-associated properties) of crypt base cells increases during differentiation and that the charge of the villus cells to which they give rise then diminishes during maturation. The charge of the bulk of the upper villus cells is the lowest of any in the intestinal cell population. The basis for the alteration in charge has not been established but the phenomenon of changing membrane surface charge (or charge-associated properties) as a function of cell differentiation, maturation and aging appears to be a general phenomenon having been found and traced in different cell populations [14, 16, 17, 28].     

Presence and histopathological effects of the Parvatrema sp. (Digenea, Gymnophallidae) in the stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae)

The stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae) has a wide geographic distribution range, including the Brazilian coasts from the northeast (Alagoas) to the south (Santa Catarina). In March 2008, an episode of mass T. plebeius mortality (70%) occurred in an intertidal bed at The Pontal da Daniela, State of Santa Catarina, Brazil. We report here high prevalences (to 100%) of the trematode parasite Parvatrema sp. Cable, 1953 (Digenea, Gymnophallidae) infecting T. plebeius at high intensities. We describe the gymnophalid, echinostomatid and unidentified metacercariae parasites infecting the clam and the host reactions elicited by them. The use of special diagnostic techniques such as Ray’s fluid thioglycollate medium (RFTM) and PCR assays to detect Perkinsus sp. pathogens, hemolymph cytology, and histopathological examinations did not show Perkinsus sp. infections, microcell infections, or neoplastic conditions. However, neither infections or pathology caused by trematode parasites; nor any other pathological condition could be uniquely correlated with the mortality event. A coincident flash flood might have contributed to cause the mortality episode. This is the first report of the Parvatrema sp. metacercariae larvae infecting the stout razor clam T. plebeius from Brazil.