Countercurrent distribution of lymphocytes from human peripheral blood in an aqueous two-phase system. II. Separation into subsets of lymphocytes with distinctive functions.
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A study was made of the effects of trypsin (0.25%) EDTA (0.02%), DMSO (10%) and suboptimal temperatures (16-18 degrees C) on the line L cells of monolayer and suspension cultures by the method of counter-current distribution in two-phase polymer system Dextran-500/poly (ethylene) glycol-6000. It has been shown that these agents cause the increase in number of cells with low partition coefficient, which is most expressed in the population of monolayer subline cells.
The distribution of two sulines of L cells, differing in adhesive properties (monolayer and suspension cultures), have been investigated using polymer two-phase system dextran-500-polyenthylenglycol-600. By counter-current distribution it has been shown that most of the cells growing in monolayer have low coefficients of partition, unlike cells grown in suspension having a high coefficient of partition. It is supposed that differences revealed in properties of the cell surface may be associated with the ability of cells to form adhesive contacts.
A study was made of the effect of cytochalazin B and cocemid on the line L cells of monolayer and suspension culture studied by the method of counter-current distribution in two-phase polymer system dextran-500 - polyethylenglycol-6000. It has been shown that cytochalazin B caused a decrease in the partition coefficient of both the subline cells irrespective of the growth phase of cell population. Colcemid decreases the partition coefficient in the log-phase of cell culture but increases it in the stationary phase. Effect of colcemid is not associated with the accumulation of metaphase cells in the cell population.
Genetic and biochemical evidence has established that a SNARE complex consisting of syntaxin 5 (Sed5)-mYkt6 (Ykt6)-GOS28 (Gos1)-GS15 (Sft1) is required for transport of proteins across the Golgi stack in animals (yeast). We have utilized quantitative immunogold labeling to establish the cis-trans distribution of the v-SNARE GS15 and the t-SNARE subunits GOS28 and syntaxin 5. Whereas the distribution of the t-SNARE is nearly even across the Golgi stack from the cis to the trans side, the v-SNARE GS15 is present in a gradient of increasing concentration toward the trans face of the stack. This contrasts with a second distinct SNARE complex, also required for intra-Golgi transport, consisting of syntaxin 5 (Sed5)-membrin (Bos1)-ERS24 (Sec22)-rBet1 (Bet1), whose v-(rBet1) and t-SNARE subunits (membrin and ERS24), progressively decrease in concentration toward the trans face. Transport within the stack therefore appears to utilize countercurrent gradients of two Golgi SNAREpins and may involve a mechanism akin to homotypic fusion.
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An inexpensive, easily constructed apparatus for mass screening of taxes of small animals with a rapid and accurate assignment of individual behavior scores has been described that should be of utility in hastening a behavioral geneticist's chores and allow him to handle more populations with fewer mazes. Also, the apparatus is well suited for use within the constraints of the undergraduate laboratory.
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Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have not found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, countercurrent distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter. These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.
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Escherichia coli mutants resistant to streptomycin exhibited differences in countercurrent distribution from the parental strains. The degree of difference from the parental strain correlated with the degree of restriction of translation and thus the particular strA allele. The changes in countercurrent distribution in the phase systems used probably resulted predominantly from surface charge alterations. The differences in countercurrent distribution in these and other mutants may be a useful selective technique to obtain different types of mutants for which specific selective techniques may not be available. In addition, it appears that the surface properties of cells, which determine their position in countercurrent distribution, are a function of the translational efficiency and fidelity, and that the surface of cells consists of a mosaic that is an expression of this translational fidelity.