Determination of surface immunoglobulin density on frozen-thawed mononuclear cells analyzed by the fluorescence-activated cell sorter

Recent data have demonstrated that differences in sIg density on B lymphocytes distinguish functionally distinct subpopulations of these cells. Other reports suggest that cyropreservation may change the frequency of sIg-bearing lymphocytes. To determine if cryopreservation alters either the frequency of sIg cells or the distribution of sIg density, PBM from normals and patients with CLL and LCL were analyzed using the FACS. Aliquots of Ficoll-Hypaque-separated PBM were controlled-rate frozen (1 °C/min) in 7.5% Me₂SO in RPMI 1640 and thawed in a 37 °C water bath on the same day. Fresh and frozen-thawed PBM aliquots were labeled with fluorescein conjugates of F(ab′) fragments of affinity chromatography-purified anti-Fab or class-specific anti-μ, anti-δ, anti-γ, or anti-α. Histograms of relative cell fluorescence, reflecting sIg density, were prepared for each aliquot with the FACS. The frequency of sIg-bearing PBM labeled with each reagent was not significantly altered by freezing. Likewise, FACS profiles demonstrated that the distribution of sIg density on normal and CLL PBM was unchanged after freezing. However, the fluorescence peak produced by frozen-thawed unlabeled cells was occasionally slightly broader than that of fresh cells, suggesting increased autofluorescence induced by freezing. These data indicate that frozen cell preparations may be utilized for the study of B-lymphocyte subsets as determined by sIg density.     

Induction of differentiation in mouse erythroleukaemia cells by the action of papain at the cell surface

The addition of one of several proteases to cultures of mouse erythroleukaemia (MEL) or human K-562 leukaemia cells can induce a substantial portion of the cells to undergo erythroid differentiation. This effect is due, at least in part, to the proteolytic action of these enzymes. The critical substrate(s) for this proteolytic action is not a component of the medium or a long-lived substance(s) released from the cells. In order to determine if the substrate(s) is located on the cell surface or intracellularly, a comparison of the ability of non-immobilized papain and immobilized papain (i.e. covalently linked to Sepharose beads which were larger than the cells) to induce MEL cell differentiation was undertaken. Both papain preparations induced the same level of differentiation. The proteolytic activity of the bead-linked papain remained associated with the beads. Therefore, proteases induce erythroid differentiation in these cells by acting proteolytically on a substrate(s) that is exterior to the cell.     

The ontogeny and distribution of B cells in normal and mutant immune-defective CBA/N mice: two-parameter analysis of surface IgM and IgD

A dual-laser fluorescence-activated cell sorter was utilized to study the distribution of the surface IgM and IgD on individual B cells of normal and immune-defective CBA/N mice. Cells from different lymphoid organs and from developing mice were studied. Two major populations of cells were seen. Those with low densities of surface IgM and intermediate-high densities of surface IgD were relatively or totally absent from the bone marrow, spleens, and lymph nodes of adult, immune-defective (CBA/N x DBA/2)F1 male mice, and developed late in ontogeny in the lymphoid organs of normal F1 female mice. By contrast, the second major population, with intermediate-high surface IgM and low surface IgD, was found in highest frequency in the lymphoid organs of immature mice, the bone marrow of adult mice, and the lymphoid organs of F1 male mice compared to F1 female mice at any age. These two major populations of B cells were further subdivided into five groups of cells to better define the surface IgM and IgD characteristics of developing B cells of immune-defective and normal mice. The relationship of these groups of cells to populations defined by other criteria are discussed.     

Intestinal cancer development in response to oral infection with high-fat diet-induced Type 2 diabetes (T2D) in collaborative cross mice under different host genetic background effects

Mammalian Genome - Type 2 diabetes (T2D) is a metabolic disease with an imbalance in blood glucose concentration. There are significant studies currently showing association between T2D and...     

THE CONSEQUENCES OF BROOD SIZE FOR BREEDING BLUE TITS. III. MEASURING THE COST OF REPRODUCTION: SURVIVAL,FUTURE FECUNDITY,AND DIFFERENTIAL DISPERSAL

To determine how the cost of reproduction varies with brood size, a population of blue tits (Parus caeruleus) breeding in Wytham Wood, England, was manipulated over a three year period. Two hundred sixteen pairs were randomly assigned 3, 6, 9, 12, or 15 nestlings; nestlings were exchanged soon after hatching. Survival of adult females (as measured by the proportion recaptured in the following winter and/or spring) declined significantly with increasing brood size in two out of three years; there was significant year-to-year variation in the relationship of recapture rate to brood size. Mean female recapture rates (averaged over the three years) declined in a linear fashion (P < 0.01). There was no significant linear or curvilinear relationship between male-recapture rate and brood size in any of the three years nor was there a significant linear or curvilinear relationship for the data averaged over the three years. Nevertheless, recapture proportions for males differed significantly with respect to brood size (χ² test, P < 0.05). The possibility that experimental brood size influences subsequent dispersal (and therefore biases measures of survival based on recapture rates to differing degrees) was examined by comparing distances moved by breeding adults from one year to the next. There was no relationship between brood size and dispersal distance within the study area for either sex, except that females given broods of three were significantly more likely to move more than 300 m than were those given broods of 6–15 young. Both males and females showed evidence of a cost with respect to future fecundity: as brood size increased, the number of surviving offspring produced in the following year decreased from 1.5–1.6 (for adults that had reared 3–6 young) to 0.4 (for those that had reared 15 young). The relationship of future reproductive success to experimental brood size did not differ among years or between the sexes. The number of eggs laid and number of young hatched in year n + 1 did not differ significantly with respect to brood size in year n; rather, differences in future fecundity reflected differences in the survival prospects of young reared in year n + 1.     

Studies on non-H-2-linked lymphocyte-activating determinants. IV. Ontogeny of the Mls product on murine B cells.

The minor lymphocyte stimulating (Mls) locus codes for lymphocyte activating determinants (LADs) on murine B lymphocytes, but not T lymphocytes. This observation was strengthened by a series of techniques which allow deletion and addition of T and B cells. These included the use of cytotoxic antisera such as anti-Thy 1.2, anti-MTLA, anti-MBLA, and complement, and the use of a goat anti-μ antisera, and finally the use of a fluorescence activated cell sorter (FACS).The studies in this report document the organ distribution and the ontogenetic appearance of the surface LADs on the surface of B lymphocytes from DBA/2N (H-2^d, Mls^a) and CBA/J (H-2^k, Mls^d) mice. Adult-like ability to stimulate H-2 identical BALB/c (H-2^d, Mls^b) and C3H/He (H-2^k, Mls^c) responder cells appeared at about 4–5 weeks of age. Inability of neonatal cells to induce an Mls-defined MLC was found not to be due to a low frequency of B lymphocytes or to the presence of suppressor cells, but due to the absence of the Mls-coded LADs on their surface. These data support the concept that the Mls-coded LADs are present on adult B lymphocytes and are specific markers of B-cell differentiation, which is preceded by membrane IgM and the δ homologue of human IgD, Ia, and the receptor for the third component of complement.     

Steady-state currents through voltage-dependent, dihydropyridine-sensitive Ca2+ channels in GH3 pituitary cells.

Ca2+ influx via voltage-dependent Ca2+ channels is known to be elicited during action potentials but possibly also occurs at the resting potential. The steady-state current through voltage-dependent Ca2+ channels and its role for the electrical activity was, therefore, investigated in pituitary GH3 cells. Applying the recently developed 'nystatin-modification' of the patch-clamp technique, most GH3 cells (18 out of 23 cells) fired spontaneous action potentials from a baseline membrane potential of 43.7 +/- 4.6 mV (mean +/- s.d., n = 23). The frequency of action potentials was stimulated about twofold by Bay K 8644 (100 nM), a Ca(2+)-channel stimulator, and action potentials were completely suppressed by the Ca(2+)-channel blocker PN 200-110 (100 nM). Voltage clamping GH3 cells at fixed potentials for several minutes and with 1 mM Ba2+ as divalent charge carrier, we observed steady-state Ca(2+)-channel currents that were dihydropyridine-sensitive and displayed a U-shaped current-voltage relation. The results strongly suggest that the observed long lasting, dihydropyridine-sensitive Ca(2+)-channel currents provide a steady-state conductivity for Ca2+ at the resting potential and are essential for the generation of action potentials in GH3 pituitary cells.