Further studies on bilitranslocase,a plasma membrane protein involved in hepatic organic anion uptake

Bilitranslocase, a plasma membrane protein involved in bilirubin and other organic anion uptake by the liver, exhibits a high molecular weight (170 000) when isolated in the presence of deoxycholate. This value is decreased to approx. 100 000 if deoxycholate is not included in the isolation medium. Both preparations can be resolved into two kinds of subunit, α and β, of 37 000 and 35 500, respectively, by reduction with 2-mercaptoethanol and addition of sodium dodecyl sulfate. Under these conditions the two subunits are still capable of high-affinity sulfobromophthalein binding and, despite the presence of the detergent, may be isolated by preparative polyacrylamide gel electrophoresis still associated with the dye. It may be suggested that the physiological subunit composition of bilitranslocase is α₂-β.     

Experimental reversal of polarity in chick embryo epiblast sheets in vitro

The dorso-ventral polarity of cells within sheets of epiblast from early chick embryos can be experimentally reversed by applying low voltages across them in experimental chambers. The induced reversal is stable and affects the position of histochemically and ultrastructurally demonstrable markers.     

Extracellular synaptic factors induce clustering of acetylcholine receptors stably expressed in fibroblasts

The clustering of nicotinic acetylcholine receptors (AChRs) is one of the first events observed during formation of the neuromuscular junction. To determine the mechanism involved in AChR clustering, we established a nonmuscle cell line (mouse fibroblast L cells) that stably expresses just one muscle-specific gene product, the AChR. We have shown that when Torpedo californica AChRs are expressed in fibroblasts, their immunological, biochemical, and electrophysiological properties all indicate that fully functional cell surface AChRs are produced. In the present study, the cell surface distribution and stability of Torpedo AChRs expressed in fibroblasts (AChR-fibroblasts) were analyzed and shown to be similar to nonclustered AChRs expressed in muscle cells. AChR-fibroblasts incubated with antibodies directed against the AChR induced the formation of small AChR microclusters (less than 0.5 micron 2) and caused an increase in the internalization rate and degradation of surface AChRs (antigenic modulation) in a manner similar to that observed in muscle cells. Two disparate sources of AChR clustering factors, extracellular matrix isolated from Torpedo electric organ and conditioned media from a rodent neuroblastoma-glioma hybrid cell line, each induced large (1-3 microns 2), stable AChR clusters with no change in the level of surface AChR expression. By exploiting the temperature-sensitive nature of Torpedo AChR assembly, we were able to demonstrate that factor-induced clusters were produced by mobilization of preexisting surface AChRs, not by directed insertion of newly synthesized AChRs. AChR clusters were never observed in the absence of extracellular synaptic factors. Our results suggest that these factors can interact directly with the AChR.     

Steroid modulation of oxytocin/vasopressin receptors in the uterus

Oxytocin (OT) and V1 vasopressin (VP) receptors are present simultaneously in several tissues, including the uterus. In myometrium these receptors mediate contractility, while in endometrium they mediate the release of other uterotonic substances as endothelin (ET). In rabbit myometrium, estrogens increase, while progesterone blunts neurohypophysial hormone receptors. However, the action of sex steroids on OT and V1 VP receptors differs in terms of the ED₅₀ and maximal effect. Therefore, at parturition, only OT receptors show a dramatic rise, while V1 VP receptors do not change, suggesting a major role for OT in labor. ET is a potent stimulator of uterine activity acting through specific receptors present on myometrial cells. These receptors as well as the endometrial localization of ET are modulated by sex steroids, indicating that ET might represent a paracrine regulator of uterine activity. In humans, OT but not V1 VP receptors increase as pregnancy progresses, confirming the primary relevance of OT in timing delivery.     

Identification and analysis of a novel human surface CD5- B lymphocyte subset producing natural antibodies.

The production of "natural" autoantibodies or antibodies, i.e., Ig that bind a variety of self- and/or exogenous Ag and arise independently of known immunization, is though to be a feature of CD5+ B lymphocytes. To determine whether other lymphocyte subsets exist that might be committed to the production of natural antibodies, human peripheral blood B cells were sorted on the basis of surface CD5 expression and differential expression of surface CD45RA (CD5+CD45RAintermediate(int), CD5-CD45RAlow(lo), and CD5-CD45RAhigh(hi)), and analyzed for the type of Ig produced after EBV infection and culture. Like their CD5+ counterparts, most CD5-CD45RAlo B lymphocytes were precursors of cells producing IgM, a major proportion of which displayed the Ag-binding features of natural antibodies. In contrast, CD5-CD45RAhi B cells comprised a high frequency of IgG-producing cell precursors, possibly including memory B lymphocytes. Six of seven IgM mAb generated from sorted CD5-CD45RAlo B cells and three of four IgM mAb from sorted CD5+ B cells were polyreactive, binding with different affinities (Kd, 10(-5) to 10(-8) M) to two or more Ag; the remaining mAb from CD5-CD45RAlo and the mAb from CD5+ B cells each bound to a single Ag (Kd, 10(-7) to 10(-8) M), beta-galactosidase and ssDNA, respectively. CD5-CD45RAlo B cells account for 4.1 +/- 1.2% (mean +/- SD in 11 healthy subjects; CD5+ B cells, 23.3 +/- 6.9%) of total B lymphocytes and display the features of quiescent cells. In a given individual, the number of CD5-CD45RAlo B cells remains constant over time. CD5-CD45RAlo and CD5+ B cells bear surface CD11b and CD14, at densities and/or frequencies apparently higher than those of CD5-CD45RAhi B lymphocytes. Despite their surface CD5- phenotype, CD45RAlo B cells express CD5+ mRNA at levels comparable with those of CD5+ B lymphocytes, whereas CD5-CD45RAhi B cells express only trace amounts of CD5 mRNA. The commitment to natural antibody production and the degree of CD5 mRNA expression suggest that the newly defined CD5-CD45RAlo B cell subset is related to CD5+ B lymphocytes, and may constitute the human homologue of the mouse Ly-1-"sister" B cell population.     

Effects of the fungicides mancozed and dithianon on mortality and reproduction of the predatory miteAmblyseius andersoni

Laboratory studies using commercial formulations of mancozeb and dithianon at concentrations equivalent to recommended field rates of 200 and 80 g/hl, respectively, were conducted to evaluate toxic effects of these fungicides on the predatory miteAmblyseius andersoni Chant. In short-term tests where females were placed on apple-leaf disks and sprayed with mancozeb, no mortality of adults was observed; however, there was a 34% decrease in fecundity, a 7.1% decrease in egg hatch, and mortality of larvae and protonymphs was 6.7%. In long-term tests, a significant reduction in fecundity was also observed. A decrease in hatch that was dependent on age at time of treatment was found when eggs were treated directly with mancozeb. No effects on mite mortality or reproduction were observed in short-term tests with dithianon. These results suggest that dithianon might be considered as a potential alternative to mancozeb for scab control.     

Identification of the peroxidation product hydroxystearic acid in Lewis lung carcinoma cells

Whole cell lipids were extracted from the Lewis lung carcinoma in vitro line C108. The fatty acids were derivatized to methylesters in order to identify endogenous oxidized derivatives by gasmass spectroscopy. The presence of 9-hydroxystearic acid and 10-hydroxystearic acid was thus evidenced for the first time in cultured mammalian cells. Moreover a linear correlation was found between the concentration of these products expressed as percentage of total fatty acid methylesters and the cell density in tissue culture flasks. This finding suggests an involvement of hydroxystearic acid in cellular functions related to the cell density in monolayer cultures.     

Developmental analysis of the cell recognition mechanism in the ciliate Euplotes raikovi

Euplotes raikovi, like other ciliates, passes through a postconjugal immaturity, operatively identified by an apparent cell inability to form mating pairs under experimental conditions that are the same as those used for inducing mating at maturity. In cells homozygous for the gene mat-2, which controls the pheromone Er-2, Er-2 mRNA synthesis and mature Er-2 secretion were shown to start from the very beginning of the life cycle and continue throughout immaturity, although to extents estimated to be 5- to 10-fold lower than at maturity. In addition, experiments of ¹²⁵ I-Er-2 binding and crosslinking provided evidence that autocrine pheromone-binding sites, showing values of the dissociation constant of the order of 10^?9 M, are on the surface of immature cells. The number of these sites per cell was estimated to increase from less than 10⁶ per cell of 5–7 fissions of age, to about 16 × 10⁶ at maturity. These results were taken to suggest that a pheromone-receptor production is stimulated during immaturity by autocrine pheromone binding to cells and that this production might be essential for the development of a pheromone-receptor density high enough to transform the cell from “immature” to “adult,” that is competent to respond as well to pheromones of conspecific, genetically different cells. © 1992 Wiley-Liss, Inc.