Active and passive cation transport and L antigen hertogeneity in low potassium sheep red cells

Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump.     

Decrease in transforming growth factor-beta binding and action during differentiation in muscle cells

We report here the effects of differentiation on the binding and action of transforming growth factor-beta (TGF-beta) in three lines of myogenic cells. In two lines (L6-A1 and C2) which irreversibly differentiate by fusing to form postmitotic myotubes, there is a virtual disappearance of TGF-beta binding sites as differentiation occurs. Analyses of the binding curves by the method of Scatchard indicates that there is little or no change in affinity but a substantial decrease in the number of binding sites. In L6-A1 cells, responsiveness to TGF-beta decreases in parallel to the loss of receptors. The decreases in TGF-beta binding and activity with differentiation are not paralleled by similar changes in another growth factor, insulin-like growth factor-I, which exhibits little change in binding and only a modest decrease in activity as L6-A1 myoblasts differentiate to form myotubes. In a third cell line (BC3H1), which exhibits reversible differentiation without fusion, there is little or no change in TGF-beta binding as the cells differentiate. Comparisons with reported decreases in binding of fibroblast and epidermal growth factors indicates that there are substantial differences in growth factor binding and actions as muscle cells differentiate, but it is not possible to make the simple generalization that differentiation is accompanied by a decrease in binding of all growth factors.     

Polyamine metabolism in enucleated mouse L-cells

The distribution of polyamines between the nucleus and the cytoplasm, and the role of the nucleus in polyamine metabolism, have been studied using cells enucleated with cytochalasin B. Spermidine and spermine were found in the nuclear and the cytoplasmic fractions of L929 cells; their concentration was 3-fold higher in the former fraction. Ornithine decarboxylase activity was only found in the cytoplasm, and this activity could be stimulated in enucleated cells by the addition of fresh medium. These cells synthesized putrescine actively, but the putrescine made was not converted to spermidine, and accumulated to relatively high concentrations. Similarly, methionine did not act as a precursor to spermidine in enucleated cells, in contrast to whole cells, although it was incorporated into cell protein. Spermidine synthesis, unlike putrescine synthesis, appears to be completely dependent on a nuclear component.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

Characterization of high-density lipoprotein binding and cholesterol efflux in cultured mouse adipose cells

Binding of high-density lipoproteins to cultured mouse Ob1771 adipose cells was studied, using labeled human HDL3, mouse HDL and apolipoprotein AI- or AII-containing liposomes. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 18, 42, 30 and 3.4 micrograms/ml, whereas the maximal binding capacities were found to be 160, 100, 90 and 21 ng/mg of cell protein. Apoprotein AI not inserted into liposomes did not bind. The binding of 125I-HDL3 was competitively inhibited by apolipoprotein AI-containing liposomes greater than mouse HDL greater than HDL3. The binding of 125I-labeled apolipoprotein AI- and 125I-labeled apolipoprotein AII-containing liposomes was competitively inhibited by HDL3, apolipoprotein AI- and apolipoprotein AII-containing liposomes. Dimyristoylphosphatidylcholine liposomes containing or not cholesterol did not interfere with the binding of labeled HDL3 or apolipoprotein-containing liposomes. Binding studies on crude membranes of Ob1771 adipose cells revealed the presence of intracellular binding sites for LDL and HDL3. Thus, adipose cells have specific binding sites for apolipoprotein E-free HDL and apolipoprotein AI (or AII) is the ligand for these binding sites. Long-term exposure of adipose cells to LDL cholesterol as a function of LDL concentration led to an accumulation of cellular unesterified cholesterol. This process was saturable and reversible as a function of time and concentration by exposure to HDL3 or apolipoprotein AI-containing liposomes, whereas apolipoprotein AII-containing liposomes did not promote any cholesterol efflux. Since long-term exposure of adipose cells to LDL and HDL3 did not affect the number of apolipoprotein B,E receptors and apolipoprotein E-free binding sites, respectively, it appears that adipose cells do not show efficient cholesterol homeostasis and thus could accumulate or mobilize unesterified cholesterol.     

Comparative studies on the determination of the A-hel blood group factor in the pig by use of various lectins]

Comparable investigations of the A-hel-receptor with Anti-AHP and 6 different phytagglutinins are reported which have been tested in 305 samples of pig blood. Anti-ADb disposing of a higher specificity is recommended for determining the Hel-factor in pigs instead of Anti-AHP.     

Dexamethasone effects on creatine kinase activity and insulin-like growth factor receptors in cultured muscle cells

We examined the effects of dexamethasone on creatine kinase (CK) activity and insulin-like growth factor I (IGF-I) binding in two skeletal muscle-derived cell lines (mouse, C2C12; rat, L6) and in one cardiac muscle-derived cell line (rat, H9c2). Dexamethasone treatment during differentiation of cultured cells caused a dose-dependent increase in CK activity as well as an increase in the degree of myotube formation in C2C12 and L6, whereas H9c2 cells did not exhibit significant CK activities during culture or dexamethasone treatment. Dexamethasone treatment of C2C12 did not stimulate proliferation in differentiating cultures, but a dose-dependent increase in the number of nuclei was observed for L6 concomitant with increased CK activity. In L6 the increased CK activity may therefore reflect a dose-dependent increase in proliferation. Short-term (48 hr) treatment of C2C12 with dexamethasone (20 nM) did not appear to alter myoblast fusion but reversibly increased CK activity. In C2C12 the observed increase in CK, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities with dexamethasone treatment suggest modulation of protein expression and/or turnover. Although the data for dexamethasone effects on CK activities varied in each of the cell lines, consistent behavior was observed in all three cell lines when IGF-I binding was examined. IGF-I binding to dexamethasone-treated cells (50 nM for 24 hr the day prior to confluence) resulted in an increased number of available binding sites, with no effect on the binding affinities. Affinity cross linking and autoradiography indicated that the increase in IGF-I binding was the result of dexamethasone up-regulation of type I IGF receptors. Our data for all three muscle cell lines suggest that similar heterologous hormone receptor modulation of type I IGF receptor sites occurs with dexamethasone treatment.     

Induction of tissue factor expression in human endothelial cells by CD40 ligand is mediated via activator protein 1, nuclear factor kappa B, and Egr-1

Induction of tissue factor expression in endothelial cells via ligation of CD40 probably figures prominently in the pathogenesis of prevalent inflammatory diseases, including atherosclerosis. However, the molecular mechanisms of tissue factor gene expression triggered by CD40 ligand (CD40L) in this cell type remain unknown. We demonstrate here that the tissue factor promoter region -278 bp to +121 bp contains the CD40L-responsive elements, consisting of activator protein 1 (AP-1)+/-, nuclear factor (NF) kappaB-, and Egr-1-binding sites. Mutations of either the AP-1- or NF-kappaB-binding sites markedly reduced the CD40L-dependent promoter activation. The AP-1 and NF-kappaB sites displayed constitutive and CD40L-enhanceable DNA binding activity, respectively. Of note, mutation of the Egr-1-binding sites, previously not associated with CD40 signaling, impaired activation of the tissue factor promoter. Accordingly, CD40L strongly induced Egr-1 protein expression and DNA binding activity to all three bindings sites. In contrast to CD40L, other established inducers of tissue factor in endothelial cells, interleukin-1beta or tumor necrosis factor alpha, did not increase the expression of Egr-1. In conclusion, induction of tissue factor gene expression in human endothelial cells by CD40L involves AP-1 and NF-kappaB as well as Egr-1, a pathway previously not implicated in CD40 signaling and distinct from that employed by certain other proinflammatory cytokines.     

Studies on glucocorticoid-induced cytolysis in cultured mouse lymphoma cells, L5178Y : I. Glucocorticoid specificity and cytoplasmic receptors in sensitive and resistant cells

1. 1. The cytolytic action of glucocorticoids such as dexamethasone was studied in a murine cultured lymphoma L5178Y cell.

2. 2. Dexamethasone-resistant cells, which could grow even in a culture medium containing 2 × 10⁻⁵ M dexamethasone, were selected from hormone-sensitive L5178Y lymphoma cells by stepwise increasing concentrations of dexamethasone in culture medium.

3. 3. The synthetic activities of macromolecules corresponded closely with the viabilities and with the resistibilities at various concentrations of dexamethasone in both cells.

4. 4. Studies of binding of dexamethasone to lymphoma L5178Y cells in vivo demonstrated that (1) equilibrium of binding at 37 °C was established within 5 min in both cells; (2) the specific binding to either whole cell or the subcellular fractions was, in sensitive cells, about double that in resistant cells.

5. 5. The cytoplasm of L5178Y lymphoma cells contains specific binding sites for dexamethasone and its binding sites (5.7 × 10⁻¹⁰ mmol/mg protein) were about 2.5-fold more than that of dexamethasone-resistant cells (2.3 × 10⁻¹⁰ mmol/mg protein).

6. 6. Studies on the dissociation constant, competition of various steroids for the specific binding with dexamethasone, and the sedimentation constant of steroid-receptor complex, suggested that the nature of cytoplasmic binding in resistant cells might be the same as that in sensitive cells.

7. 7. The nuclear binding of dexamethasone was extremely dependent on the affinity of cytoplasm to steroid. Studies suggested an equal level of nuclear association sites for cytoplasmic steroidreceptor complex in the two cell types.

     

雌激素对兔子宫内膜细胞EGF受体和细胞周期的调控

 由受体放射配基结合分析证明家兔子宫内膜细胞的EGF受体Kd值为0.53nmol/L,每个细胞的最大结合容量为1.11×10~4结合位点。10~(-10)mol/L雌二醇处理24h,细胞的最大结合容量增至2.75×10~4结合位点数/细胞,而Kd值无明显变化,可是,当10~(-5)mol/L雌二醇处理24h,细胞的EGF受体结合率,DNA合成速度率均下降。G_0/G_1期细胞比值明显下降,而G_2+M期和S期细胞明显上升。     

Integrin and neurocan binding to L1 involves distinct Ig domains.

The cell adhesion molecule L1, a 200-220-kDa type I membrane glycoprotein of the Ig superfamily, mediates many neuronal processes. Originally studied in the nervous system, L1 is expressed by hematopoietic and many epithelial cells, suggesting a more expanded role. L1 supports homophilic L1-L1 and integrin-mediated cell binding and can also bind with high affinity to the neural proteoglycan neurocan; however, the binding site is unknown. We have dissected the L1 molecule and investigated the cell binding ability of Ig domains 1 and 6. We report that RGD sites in domain 6 support alpha5beta1- or alphavbeta3-mediated integrin binding and that both RGD sites are essential. Cooperation of RGD sites with neighboring domains are necessary for alpha(5)beta(1). A T cell hybridoma and activated T cells could bind to L1 in the absence of RGDs. This binding was supported by Ig domain 1 and mediated by cell surface-exposed neurocan. Lymphoid and brain-derived neurocan were structurally similar. We also present evidence that a fusion protein of the Ig 1-like domain of L1 can bind to recombinant neurocan. Our results support the notion that L1 provides distinct cell binding sites that may serve in cell-cell or cell-matrix interactions.     

The transition from HK to LK phenotype in the red cells of newborn genetically LK lambs

Red cells from newborn lambs were separated into different age populations by centrifugation, and cells with fetal hemoglobin (Hb) were distinguished from those with adult Hb by an acid elution technique. Changes were followed during development in rates of K+ transport (active and passive), numbers of Na+/K+ pump sites per cell, cell volumes, and numbers of Lp and L1 antigen sites per cell. These changes were correlated with the percentage of cells with adult hemoglobin. (The Lp and L1 antigens are associated with K+ transport in that specific alloantibody against Lp, anti-Lp, stimulates active transport, and anti-L1 inhibits passive transport.) Active K+ transport decreased during development because of a decline in number of Na+/K+ pumps (from measurements of ouabain binding) and because of an alteration in the affinity of the pumps for intracellular K+ (from kinetic studies in which the intracellular K+ concentration was varied). Cells with fetal Hb had fewer Lp sites and were larger than cells with adult Hb. As transport properties changed, the number of Lp sites increased and continued to increase after all the cells had adult Hb Cells with fetal Hb had as many L1 sites as lamb cells with adult Hb, but the number of L1 sites was less than those found previously for adult sheep. A population of small cells with intermediate K+ concentration and intermediate numbers of Lp sites appeared soon after birth. The various points of evidence suggested that the developmental process leading to cells with adult transport properties was a gradual one and did not coincide precisely with the switch from fetal to adult Hb.     

Binding of Escherichia coli lipopolysaccharide to fasciculata-reticularis and glomerulosa cells evaluated by flow cytometry

Binding of Escherichia coli lipopolysaccharide (LPS) to the two cell types of the adrenal cortex: fasciculata-reticularis and glomerulosa cells has been studied by flow cytometry and using fluorescein-labeled lipopolysaccharide (FITC-LPS). The binding characteristics were different in relation to time course and number of binding sites. Both fasciculata-reticularis and glomerulosa cells bound LPS in a specific and saturable process. Fasciculata-reticularis cells showed a higher affinity for LPS binding than glomerulosa cells as deduced from Hill plots. Unlabeled LPS decreased FITC-LPS binding in both fasciculata-reticularis and glomerulosa cells, suggesting competition of both ligands for a limited number of binding sites. Lipid A seemed not to be essential for binding of LPS to fasciculata-reticularis cells. However, serum constituents inhibited FITC-LPS binding to both cell types, possibly due to cell interaction with HDL. The exposure of cells to LPS during cell culture did not modify the number of binding sites, but revealed cell size and surfaces structure changes.     

Specific binding sites for a parvovirus, minute virus of mice, on cultured mouse cells.

The early interactions between parvoviruses and host cells have not been extensively described previously. In this study we have characterized some aspects of viral binding to the cell surface and demonstrated the existence of specific cellular receptor sites for minute virus of mice (MVM) on two murine cell lines that are permissive for viral growth. The interaction had a pH optimum of 7.0 to 7.2, and both the rate and extent of the reactions were slightly affected by temperature. Mouse A-9 cells (L-cell derivative) had approximately 5 X 10(5) specific MVM binding sites per cell, and Friend erythroleukemia cells had 1.5 X 10(5) MVM sites per cell. In contrast, the nonpermissive mouse lymphoid cell line L1210 lacked specific viral receptors. Also, cloned lines of A-9 cells resistant to viral infection have been isolated. One of these lines lacked the "specific" virus attachment sites but exhibited low levels of nonsaturable virus binding. Based on these examples, infectivity is correlated with the presence of specific viral receptors on the cell surface.     

Interactions of agonists and antagonists with beta-adrenergic receptors on intact L6 muscle cells

A binding assay has been developed to characterize beta-adrenergic receptors on intact L6 muscle cells. The affinity of beta-adrenergic receptors for the radioligand iodohydroxybenzylpindolol (IHYP) was the same in membrane preparations and in intact cells when determined by either equilibrium binding or kinetic analysis. The number of specific IHYP binding sites per cell was approximately the same on intact cells as on membranes. The pharmacological properties of antagonists indicated that the receptors on intact cells were identical to those on membranes. However, the beta-adrenergic receptors on intact cells had a 100-400 fold lower affinity at equilibrium for the agonist isoproterenol than did beta-adrenergic receptors on membranes. This low affinity of the receptor for agonists as measured by inhibition of radioligand binding in intact cells has also been observed in C6 (2) and S49 (3) cells. Our results suggest that beta receptors on intact cells after a 1 minute incubation was similar to the KD value for isoproterenol measured in membranes at equilibrium in the presence of GTP. After 1-2 minutes of exposure to a low concentration of agonist, binding of IHYP was no longer inhibited. These results suggest that agonists rapidly convert the beta receptors on intact cells to a state which has a low affinity for agonists. The affinity of the receptor for antagonists did not change during the incubation.     

Internal binding sites for MSH: analyses in wild-type and variant Cloudman melanoma cells

Cloudman S91 mouse melanoma cells express both external (plasma membrane) and internal binding sites for MSH. Using 125I-beta melanotropin (beta-MSH) as a probe, we report here an extensive series of studies on the biological relevance of these internal sites. Cells were swollen in a hypotonic buffer and lysed, and a particulate fraction was prepared by high-speed centrifugation. This fraction was incubated with 125I-beta-MSH with or without excess nonradioactive beta-MSH in the cold for 2 hours. The material was then layered onto a step-wise sucrose gradient (8-80%) and centrifuged (156,000g, 60 min); fractions were collected and counted in a gamma counter or assayed for various enzymatic activities. The following points were established: 1) Specific binding sites for MSH were observed sedimenting at an average density of 50% sucrose in amelanotic cells and at higher densities in melanotic cells. 2) These sites were similar in density to those observed when intact cells were labeled externally with 125I-beta-MSH and then warmed to promote internalization of the hormone. 3) Most of the internal binding sites were not as dense as fully melanized melanosomes. 4) In control experiments, the MSH binding sites were not found in cultured hepatoma cells. 5) Variant melanoma cells, which differed from the wild-type in their responses to MSH, had reduced expression of internal binding sites even though their ability to bind MSH to the outer cell surface appeared normal. (MSH-induced responses included changes in tyrosinase, dopa oxidase, and dopachrome conversion factor activities, melanization, proliferation, and morphology.) 6) Isobutylmethylxanthine, which enhanced cellular responsiveness to MSH, also enhanced expression of internal binding sites. The results indicate that expression of internal binding sites for MSH is an important criterion for cellular responsiveness to the hormone.     

The mechanism of gamma-secretase: multiple inhibitor binding sites for transition state analogs and small molecule inhibitors

Transition state analogs pepstatin methylester (PME) and L685458 have been shown to inhibit gamma-secretase non-competitively (Tian, G., Sobotka-Briner, C., Zysk, J., Liu, X., Birr, C., Sylvester, M. A., Edwards, P. D., Scott, C. W., and Greenberg, B. D. (2002) J. Biol. Chem. 277, 31499-31505). This unusual kinetics suggests physical separation of the sites for substrate binding and catalysis with binding of the transition state analogs to the catalytic site and not to the substrate binding site. Methods of inhibitor cross-competition kinetics and competition ligand binding were utilized to address whether non-transition state small molecule inhibitors, which also display non-competitive inhibition of gamma-secretase, inhibit the enzyme by binding to the catalytic site as well. Inhibitor cross-competition kinetics indicated competitive binding between the transition state analogs PME and L685458 and between small molecules arylsulfonamides and benzodiazepines, but non-competitive binding between the transition state analogs and the small molecule inhibitors. These results were indicative of two inhibitor binding sites, one for transition state analogs and the other for non-transition state small molecule inhibitors. The presence of two inhibitor binding sites for two different classes of inhibitors was corroborated by results from competition ligand binding using [3H]L685458 as the radioligand. Although L685458 and PME displaced the radioligand at the same concentrations as for enzyme inhibition, arylsulfonamides and benzodiazepines did not displace the radioligand at their Ki values, a result consistent with the presence of two inhibitor binding sites. These findings provide useful insights into the catalytic and regulatory mechanisms of gamma-secretase that may facilitate the design of novel gamma-secretase inhibitors.     

Binding characteristics of M and L isoantibodies to high and low potassium sheep red cells.

Binding of highly purified 125I labeled M and L antibodies, both belonging to the immunoglobulin G class, was studied in high potassium (HK) and low potassium (LK) sheep red cells. Anti-M and anti-L bound specifically to M and L antigen positive HK and LK red cells, respectively. Nonspecific binding was higher for anti-L to HK cells than for anti-M to LK cells. Once bound, the M and L antibodies were capable of inducing complement dependent immune hemolysis. Only 75-100 and 500-750 molecules of anti-M and anti-L immunoglobulins were required to hemolyze 50% of HK (MM) and LK (LL) red cells, respectively, suggesting that the M and L antigens may be clustered on the surfaces of these cells. Equilibrium binding studies revealed that the maximum number of M sites is 3-6 x 10(3) in HK (MM) and 1.5-4 x 10(3) in LK (LM) cells, respectively. In comparison, the number of L antigens is slightly lower in LK cells, about 1.2-1.8 x 10(3) in LL and less in LM(LK) red cells. The number of M and L antigens, therefore, is more than an order of magnitude larger than that of the Na+K+ pumps measured previously in these cells by 3H-ouabain binding, thus precluding a quantitative correlation between M and L antigens and the Na+K+ pumps different in the three genetic types of sheep red cells. The binding affinities of both anti-M and anti-L could not be described by a single equilibrium dissociation constant indicating heterogeneous antibody populations and /or variability in the antigenic sets of individual HK or LK cells. The pronounced heterogeneity of antigens and/or antibodies in both the M and L systems was reflected in the antibody association kinetics, which also exhibited a remarkable temperature dependence. The data suggest that the correlation between the M and L antigens and the Na+K+ pump molecules is more complex than that in goat red cells previously reported by others.