Sequential changes in glycolipid expression during human B cell differentiation: enzymatic bases

We have previously reported that human B cell differentiation is accompanied by sequential changes in glycosphingolipid expression. Pre-B cells contain lacto-series type II chain-based glycolipids and GM3 ganglioside; mature/activated B cells do not synthesize lacto-series compounds but express GM3 and globo-series glycolipids (Gb3 and Gb4); terminally differentiated B cells, in addition to these compounds, also contain GM2 ganglioside. At the cell surface, Gb3, Gb4 and GM2 constitute stage-specific antigens. To elucidate the biosynthetic mechanism leading to these modifications we have compared activities of the glycosyltransferases involved in the core structure assembly and the first elongation steps of neo-lacto, ganglio- and globo-series glycolipids. These glycosyltransferase activities have been measured in B cell lines and normal B lymphocytes at various stages of differentiation. We first determined the optimal requirements of the four glycosyltransferases which wynthesize Lc3, GM3, Gb4 and GM2 glycolipids in B lymphocytes and then tested these enzymes and the Gb3 synthetase in the selected B cells. The following results were obtained: ß1 → 3N-Acetylglucosaminyltransferase (Lc3 synthetase) has a high activity in pro- and pre-B cells whereas it is undetectable in more differentiated cells; α2 → 3 sialyltransferase (GM3 synthetase) is activated from the pre-B cell stage to the terminally differentiated myeloma cells; α → 4 galactosyltransferase (Gb3 synthetase) is only detected in cells representing the late stages of B cell differentiation; ß1 → 3N-Acetylgalactosaminyltransferase (Gb4 synthetase) is only found in some lymphoblastoid cell lines, representative of activated B cells whereas the ß1 → 4 N-Acetylgalactosaminyltransferase (GM2 synthetase) has a high activity in these lymphoblastoid cell lines and in terminally differentiated myeloma cells. These results suggest that the sequential shifts in the three major glycosphingolipid series observed during B cell differentiation are mostly due to sequential activations of the corresponding glycosyltransferases.     

GM2 Ganglioside Regulates the Function of Ciliary Neurotrophic Factor Receptor in Murine Immortalized Motor Neuron-Like Cells (NSC-34)

We previously reported that ciliary neurotrophic factor (CNTF) increased the serum-free cell survival of immortalized motor neuron-like cells (NSC-34), and addition of the exogenous ganglioside GalNAc4(Neu5Ac3)Gal4GlcCer (GM2) facilitated cell survival together with CNTF. Moreover 1,4 N-acetylgalactosaminyltransferase (GM2 synthase) activity increased in NSC-34 cells cultured with CNTF. We now have examined whether CNTF-induced cell survival is associated with the collaboration between GM2 and the CNTF receptor (CNTF-R). Despite the presence of CNTF (50 ng/ml), anti-CNTF-R antibody caused cell death and prevented the up-regulation of GM2 synthase expression. The addition of GM2 (1 to 20 M) abrogated the anti-CNTF-R antibody effect which shortened cell survival and blocked GM2 synthase activation. Use of [¹²⁵I]CNTF showed the specificity of CNTF binding in NSC-34 cells in situ. GM2 produced a 5-fold increase in the CNTF binding affinity per cell but did not change the binding site number. The study by metabolic labeling with [1–¹⁴C]N-acetyl-D-galactosamine ([¹⁴C]GalNAc) showed that biosynthesized GM2 was involved in the immunoprecipitation of CNTF-R. These findings indicate that up-regulated GM2 synthesis induces functional conversion of CNTF-R to the activated state, in which it has affinity for CNTF. We conclude that GM2 is a bio-regulating molecule of CNTF-R in motor neurons.     

Influence of Gangliosides on the IL-2– and IL-4–Dependent Cell Proliferation

Ganglioside-induced apoptosis in the cells of IL-2–dependent cytotoxic murine cell line CTLL-2 was shown to be caspase dependent: GM1-, GM2-, and GD3-induced suppression of cell proliferation was cancelled by a general caspase inhibitor Z-VAD-FMK. Ganglioside-induced apoptosis pathways are different for different individual glycolipids; the differences exist both at the initiation and effector stages of the caspase cascade. Only for GM1-induced process, molecular mechanisms of signal transduction coincide with the ones for CD95 and TNF: the participation of both the main initiation caspases 8, 1, and 4, and caspases 3 and 9 as well, has been shown. Caspase 3 participates in the pathway induced by GM3, GD1a, GD1b, and GT1b, but not by GM2. As morphological features show, tumor-associated ganglioside GM2 is also a stimulus of programmed cell death (PCD) for CTLL-2 cell line: addition of GM2 into cell culture has resulted in appearance of annexin V-positive cells and in accumulation of DNA breaks (shown by the TUNEL direct dyeing of the open ends). But a caspase 3 inhibitor Z-DEVD-FMK did not restore the cell proliferation suppressed by GM2, and addition of a fluorescent substrate of caspase 3 Ac-DEVD-AFC did not result in the fluorescence development. So caspase 3 does not participate in downstream pathways of GM2-induced cell apoptosis, and a PCD-effector system other than the apoptosome-mediated one is involved here.     

Effect of gangliosides on activation of the alternative pathway of human complement

Liposomes as defined model membranes were used to quantitatively study the effects of specific sialic acid containing glycolipids on activation of the alternative pathway of human C. Liposomes containing dimyristoylphosphatidylethanolamine, cholesterol, and cerebrosides at molar ratios of 1.0/0.75/0.33 activated the alternative pathway in human serum treated with MgEGTA. Activation was measured by C3 conversion and the deposition of total C3 and functional C3b on the liposome surface. The monosialoganglioside GM1, when incorporated into the activating liposome membrane at molar ratios between 10(-5) and 10(-2), inhibited activation in a dose-dependent manner. Sialosylparagloboside also inhibited activation in human serum, and inhibition was completely reversed after neuraminidase treatment. The degree of inhibition by GM1 correlated with the relative amount of GM1 exposed on the liposome surface. Sialic acid did not directly inhibit the binding of C3b when liposomes containing gangliosides were incubated with the purified components C3, B, D, and P. GM1 did inhibit activation when liposomes were incubated with a mixture of purified C3, B, D, P, H, and I. Binding assays with radiolabeled H showed increased binding of H to liposome-bound C3b in the presence of GM1. These results establish the ability of sialic acid on glycolipids to promote H binding to C3b and thereby regulate alternative pathway activation on a defined lipid membrane.     

Surface activation of the cell adhesion fragment of fibronectin

One of the earliest events in the adhesion of fibroblasts to a substratum is the recognition by the cells of macromolecular adhesive factors, such as fibronectin. This early event is followed by a complex series of cell alterations leading to adhesion and spreading. To identify cell surface components involved in the initial cell-fibronectin recognition step, we have employed an assay involving latex particles coated with radiolabelled plasma Fibronectin (Fn). In previous studies from this laboratory (Harper & Juliano , J cell biol 87 (1980) 755) [28], we demonstrated that Fn-mediated adhesion of CHO cells is temperature-dependent, cation-dependent and sensitive to cytoskeletal disrupting agents; by contrast, binding of 3H-Fn beads was unaffected by these factors, indicating that this process reflects binding and recognition events at the cell surface which are independent of cytoskeletal and metabolic activity. Biological specificity of 3H-Fn bead-to-cell binding was confirmed by the ability of anti-Fn antisera to completely block the process. To examine surface components which may mediate binding we treated Fn beads with purified glycosaminoglycans (GAGs) or glycolipids prior to incubation with cells. Among the GAGs tested, heparin, heparan sulfate and dermatan sulfate blocked bead binding in a dose-related fashion with heparin being most potent. The gangliosides GT1, and GM1, also inhibited bead binding. However, treatment of cells with neuraminidase had no effect on bead binding while subsequent analysis of treated cells by thin layer chromatography revealed a drastic reduction in the amount of GM3, the predominant CHO cell ganglioside. CHO cells were also incubated with a panel of proteolytic enzymes to study the possible role of cell surface proteins or glycoproteins in Fn bead binding. We found 3H-Fn bead binding to be quite sensitive to pretreatment with thermolysin, pronase, and papain but only moderately sensitive to treatment with trypsin. From our findings we suggest: (1) binding of Fn beads to CHO cells reflects an early step in the adhesion process; (2) glycolipids may block bead binding but are probably not the endogenous binding site for Fn; (3) protease sensitive components (glycoproteins or proteoglycans) may be more likely candidates as cell surface-binding sites for Fn.     

The Ly-10 antigen is a marker of mouse-activated T lymphocytes

Ly-10.1 is a lymphocyte surface antigen controlled by a gene linked to the Ly-1.1 locus and expressed on activated T helper, T suppressor (Ts), and cytotoxic T lymphocytes (CTL). In this report, we describe the following:

Simultaneous expression by porcine aorta endothelial cells of glycosphingolipids bearing the major epitope for human xenoreactive antibodies (Galα1–3Gal), blood group H determinant andN-glycolylneuraminic acid

Glycosphingolipids were isolated from primary cultures of porcine endothelial cells labelled with¹⁴C-galactose or¹⁴C-glucosamine. They were characterized by their mobility on thin layer chromatogram, their sensitivity to exoglycosidases, and their labelling with antibodies. In addition to the major glycosphingolipids, globotetra-and globotriaosylceramide, minor ones were identified as penta-and heptaglycosylceramide of the neolactoseries terminated by either Gal1–3Gal-(xenoreactive epitope) or Fuc1–2Gal-(H determinant). Two gangliosides were found, GM3 and GD3, andN-glycolylneuraminic acid was their major sialic acid. Therefore, porcine endothelial cells differ from human endothelial cells by expression of glycosphingolipids that are absent in man: two Gal1–3Gal-terminated glycolipids recognized by human natural antibodies, and twoN-glycolylneuraminic acid-terminated gangliosides which are potent immunogens.Abbreviations HPTLC high performance thin-layer chromatography - GSL glycosphingolipid - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - PAEC porcine aorta endothelial cell     

Mechanisms of transmembrane signalling in human T cell activation

Several monoclonal antibodies directed against a number of T cell surface molecules are used to elucidate the role of these molecules (cell surface molecules) in T cell activation. The activation of T cells via these molecules are both antigen-dependent (CD3/TcR complex) and antigen-independent. Irrespective of their antigen-dependency, these monoclonal antibodies activate T cells by a classical signal transduction pathway, in which the binding of monoclonal antibodies to their cell surface receptors leads to activation of phospholipase C resulting in the the depolarization of plasma membrane, hydrolysis of IP2 and IP3 and DAG, the second messengers. IP3 leads to mobilization of intracellular calcium to contribute to an increase in [Ca⁺⁺]_i, whereas DAG causes activation and translocation of PKC and an increasing apparent affinity for Ca⁺⁺. The role of IN in the mobilization of intracellular calcium is emerging. In addition, influx of extracellular calcium also contributes to increase in [Ca^–+];. The increase in [Ca⁺⁺]; following activation via some T cell surface antigen is predominantly due to intracellular mobilization of Ca^–+ (e.g. CD3/TcR complex), whereas activation via other T cell surface antigen, the increase in [Ca^+–]_i is almost entirely due to an influx of extracellular calcium (e.g. CD5 antigen). All these molecules activate autocrine system of T cell growth, namely IL-2 production, IL-2 receptor expression and T cell proliferation.     

Studies on macrophage-activating factor (MAF) in antitumor immune responses

Summary In the present study we investigated some of the physicochemical properties of macrophage-activating factor(s) (MAF) produced by the tumor-immune Lyt-1⁺2^– T cell subset. Supernatant from mixed culture of spleen and lymph node cells, obtained from C3H/HeN mice immunized with syngeneic MH134 hepatoma or MCH-1-A1 fibrosarcoma, with the corresponding tumor cells exhibited the capability of activating peritoneal exudate macrophages to exert their cytostatic and cytolytic activities on tumor cells. Such MAF production was abolished by treatment of tumor-immune spleen and lymph node cells with anti-Thy-1.2 or anti-Lyt-1.1 antibody plus complement (C) before culturing. Anti-Lyt-2.1 and/or anti-asialo GM1 plus C treatment, however, had only marginal effect on the generation of MAF by these cells, despite the complete disappearance of natural killer (NK) cell activity of spleen and lymph node cells after the treatment with anti-asialo GM1 plus C. Thus, the tumor-specific Lyt-1⁺2^– T cell subset could fulfill a crucial role in generating MAF without the support of NK cells. The MAF activity was heat, acid, and trypsin sensitive. On Sephacryl S-300 column, MAF activity was eluated in a broad single peak around a molecular weight (m.w.) of 70,000 daltons. Antiviral activity was detected in the concentrated pool of MAF-containing fractions from Sephacryl S-300. Gel permeation analysis using HPLC also showed a coincident peak of MAF and antiviral activities at a m.w. of approximately 70,000 daltons. In addition, MAF activity was almost completely neutralized by incubation with rabbit antiserum against recombinant murine -interferon (IFN). Taken together, these results indicate that MAF generated by tumor-immune Lyt-1⁺2^– T cell subset is closely related to IFN.     

Brefeldin A induced inhibition of de novo globo- and neolacto-series glycolipid core chain biosynthesis in human cells. Evidence for an effect on beta 1-->4galactosyltransferase activity.

De novo synthesis of neolacto-series glycolipids has been studied in human cell lines via metabolic labeling of ceramide with [3H]serine. Intense labeling of ceramide mono- and dihexoside glycolipids occurred with labeling of progressively longer chain derivatives with increasing time. Most of the label was recovered in neutral glycolipids with about 5% of the total labeling in the ganglioside fraction. Experiments done using cell treatment with 2.5 micrograms/ml brefeldin A resulted in a stimulation in the total amount of labeling, accumulation of a neutral glycolipid identified as Lc3 due to inhibited transfer of the neolacto-series core chain terminal beta-Gal residue, and a corresponding inhibition of labeling of longer chain neutral glycolipids in all cell lines. Brefeldin A also blocked synthesis of the globo-series precursor, Gb3, longer chain sialylated structures such as IV3NeuAcnLc4, but not de novo GM3 synthesis. Brefeldin A treatment had no effect on cellular beta 1-->3N-acetylglucosaminyl-, beta 1-->4galactosyl-, or alpha 1-->3fucosyltransferase specific activities, nor was it inhibitory in beta 1-->4galactosyltransferase assays in vitro. The results describe brefeldin A-induced blocks in globo- and neolacto-series glycolipid biosynthesis, consistent with differential localization of enzymes in intracellular membranes. In particular, the results suggest that the beta 1-->4galactosyltransferase in these cells is either not redistributed by brefeldin A or is otherwise rendered nonfunctional.     

Blocked ricin-conjugated T cell immunotoxins: Effect of anti-CD6-blocked ricin on normal T cell function

Summary The biological properties of an immunotoxin composed of an anti-CD6 monoclonal antibody conjugated to whole ricin, which had been modified so that the galactose-binding sites of the B chain were blocked (blocked ricin), were examined. Treatment of peripheral blood lymphocytes with anti-CD6-blocked ricin for a 24-h period prevented T cell proliferation induced by phytohemagglutinin in a dose-dependent manner with concentrations causing 50% inhibition (IC₅₀) ranging from 5 pM to 30 pM. In contrast, treatment with either blocked ricin alone or with a control immunotoxin prepared with a B-cell-lineage-restricted monoclonal antibody gave IC₅₀ values of approximately 2 nM. Although shortening the duration of the anti-CD6-blocked ricin treatment to as little as 3 h had little significant effect on the observed inhibition, T cell viability experiments demonstrated that the magnitude of immunotoxin-induced killing after a given time period is significantly higher when the target cells become activated. Thus, from the initial concentration of cells treated with anti-CD6-blocked ricin placed in culture, 40%–45% viable cells remained after 2 days yet only 3%–9% remained if phorbol ester and Ca²⁺ ionophore were added; activation of T cells after mock treatment using blocked ricin plus nonconjugated anti-CD6 demonstrated that this effect was not the result of activation alone. The toxicity of anti-CD6-blocked ricin was also measured by inhibition of PHA-induced clonogenic growth of normal T cells. Continuous treatment of the cells using anti-CD6-blocked ricin at 0.1 nM resulted in a surviving fraction of about 3.5 × 10^–3; when immunotoxin treatment was for 24 h or less, the surviving fraction was only about 10^–1. As an indication of the unique specificity of anti-CD6-blocked ricin, immunotoxin pretreatment of potential responder cells prevented the generation of allogeneic cytolytic T lymphocytes in mixed lymphocyte cultures yet had little effect on the generation of interleukin-2-induced lymphokine-activated killer cell activity. We conclude that anti-CD6-blocked ricin demonstrates a cellular specificity and potency that make it a highly promising anti-T cell reagent.     

Polyclonal activation of human lymphocytes and induction of cytotoxic lymphocytes by streptococcal preparations

Polyclonal activation of human peripheral blood lymphocytes (PBLs)in vitro by preparations ofStreptococcus pyogenes Su strain (OK-432) and other heat-killed strains was investigated. The streptococcal preparations tested induce a proliferative response of PBLs via interleukin-2 (IL-2)-independent pathways. The proliferative response is accompanied by the generation of lymphoblastic cells (LBCs), which consist of heterologous lymphocyte populations: CD4⁺ helper type of T cells, and CD4^–CD8^– double-negative (DN) lymphocytes, including both CD3⁺ TcR ⁺ T cells and CD2⁺CD3^– immature type of T or non-T cell type of lymphocytes. Almost all the LBCs express Leu19, TfR (transferrin receptor), LFA-1 and CD38 (OKT10) antigens, which are expressed on activated T cells, NK cells and some other lymphocytes. The proliferative response of human PBLs is also accompanied by the generation of potent cytotoxic activity against NK-sensitive and -resistant targets. C-dependent cytolysis and cell sorting experiments of OK-432-activated LBCs revealed that both CD3⁺ and CD3^– types of CD4^–CD8^– DN lymphocytes, but not CD4⁺ helper T cells, may be major populations responsible for the cytotoxicity induced. On the other hand, CD4^–CD8 T cells may be required for the proliferation of PBLs and generation of cytotoxic effector cells. These results suggest that the OK-432 and other streptococcal preparations stimulate the human PBLsin vitro to induce the proliferation/activation of CD4⁺ T cells, mediating the following generation of DN cytotoxic effector lymphocytes.     

A function for tyrosine phosphorylation of type 1 inositol 1,4,5-trisphosphate receptor in lymphocyte activation

Sustained elevation of intracellular calcium by Ca²⁺ release–activated Ca²⁺ channels is required for lymphocyte activation. Sustained Ca²⁺ entry requires endoplasmic reticulum (ER) Ca²⁺ depletion and prolonged activation of inositol 1,4,5-trisphosphate receptor (IP₃R)/Ca²⁺ release channels. However, a major isoform in lymphocyte ER, IP3R1, is inhibited by elevated levels of cytosolic Ca²⁺, and the mechanism that enables the prolonged activation of IP₃R1 required for lymphocyte activation is unclear. We show that IP₃R1 binds to the scaffolding protein linker of activated T cells and colocalizes with the T cell receptor during activation, resulting in persistent phosphorylation of IP₃R1 at Tyr353. This phosphorylation increases the sensitivity of the channel to activation by IP₃ and renders the channel less sensitive to Ca²⁺-induced inactivation. Expression of a mutant IP3R1-Y353F channel in lymphocytes causes defective Ca²⁺ signaling and decreased nuclear factor of activated T cells activation. Thus, tyrosine phosphorylation of IP3R1-Y353 may have an important function in maintaining elevated cytosolic Ca²⁺ levels during lymphocyte activation.     

Effect of gemcitabine on immune cells in subjects with adenocarcinoma of the pancreas

Effects of gemcitabine (Gemzar) on immune cells were examined in pancreas cancer patients to determine whether it was immunosuppressive, or potentially could be combined with vaccines or other immunotherapy to enhance patients responses to their tumors. Blood was obtained at five time-points, before therapy, 3–4 days after initial gemcitabine infusion and immediately preceding three additional weekly infusions. Effects on T-cell subsets, B-cells, myeloid dendritic cell precursors, antigen presenting cells (APC), activated/memory, and naive cells were examined. Functional activity was measured by intracellular staining for cytokines before and after T-cell activation, and by interferon production in EliSpot responses to tumor presentation. Although absolute lymphocyte counts decreased with the initial treatment with gemcitabine infusion, the counts stabilized during subsequent treatments, then returned within normal ranges seven days after the fourth treatment so that the absolute lymphocyte count no longer differed significantly from that prior to treatment. These effects on absolute lymphocyte counts were mirrored by statistically significant decreases in absolute numbers of CD3 and CD20 lymphocytes during these time periods. The proportions of T and B-cells, however did not change significantly with therapy, although significance changes were observed in some specialized subsets. A decrease in the proportions of the major BDCA-1⁺, CD1b myeloid dendritic cell subset and a reciprocal increase in the minor BDCA-3⁺ dendritic cell subsets resulted at 3–4 days, then their levels returned to normal. No significant changes in percentages of CD86 and CD80 APCs or CD4⁺, CD25⁺T-cells were documented. Increased percentages of CD3⁺, CD45RO⁺ memory lymphocytes reached significance at day 7, then declined to statistically significant decrease at days 14 and 21 after the second and third infusions, respectively. Immune T-cells were functional in pancreas cancer patients treated with gemcitabine. The data suggest that gemcitabine therapy may decrease memory T-cells and promote naive T-cell activation. We conclude that gemcitabine therapy (1) is not immunosuppressive and (2) may enhance responses to specific vaccines or immunotherapy administered to activate or support immune responses directed toward driving effector immunity to cancer cells.     

Signal role for activation of caspase-3-like protease and burst of superoxide anions during Ce<Superscript>4+</Superscript>-induced apoptosis of cultured <Emphasis Type="Italic">Taxus cuspidata</Emphasis> cells

The signal events of 1 mM Ce⁴⁺ (Ce(NH₄)₂(NO₃)₆)-induced apoptosis of cultured Taxus cuspidata cells were investigated. The percentage of apoptotic cells increased from 0.82% to 51.32% within 6 days. Caspase-3-like protease activity became notable during the second day of Ce⁴⁺-treatment, and the maximum activity was 5-fold higher than that of control cells at the fourth day. When the experiment system was pretreated with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) at 100 M, caspase-3-like activity resulted in distinct inhibition by 70% and 77.3% after 3 and 4 days of induction. Furthermore, 100 M Ac-DEVD-CHO partially reduced the apoptotic cells by 58.6% and 60.8% at day 4 and 5 respectively. Ce⁴⁺ induced superoxide anions (O₂·–) transient burst, and the first peak appeared at around 3.7–4 h, the second appeared at about 7 h. Both O₂·– burst and cell apoptosis were effectively suppressed by application of diphenyl iodonium (NADPH oxidase inhibitor). Inhibition of O₂·– production attenuated caspase-3-like activation by 49% and 53.6% during day 3 and 4 respectively. In addition, a total of 15 protein spots changed in response to caspase-3-like protease activation were identified by two-dimensional gel electrophoresis. These results suggest that Ce⁴⁺ of 1 mM induces apoptosis in suspension cultures of T. cuspidata through O₂·– burst as well as caspase-3-like protease activation. The burst of O₂·– exerts its activity as an upstream of caspase-3-like activation. Our results also implicate that other signal pathways independent of an O₂·– burst possibly participate in mediating caspase-3-like protease activation.     

Ganglioside GM1 Activates the Mitogen-Activated Protein Kinase Erk2 and p70 S6 Kinase in U-1242 MG Human Glioma Cells

Abstract: Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U-1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [³H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U-1242 MG cells with GM1 caused activation of the mitogen-activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1-stimulated Erk2 activation and GM1-stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70^s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1-stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet-derived growth factor also activated both Erk2 and p70^s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1-mediated DNA synthesis.     

Light activates binding of membrane proteins to chloroplast RNAs in Chlamydomonas reinhardtii

Several membrane proteins were previously shown to bind to the 5 leader of the chloroplast psbC mRNA in the unicellular eukaryotic alga Chlamydomonas reinhardtii. This study showed that these proteins have affinity for AU-rich RNAs, as determined by competition experiments. In addition, their binding activities are enhanced 13–15-fold by light, and a 46 kDa protein is activated within 1–10 min. This activation could be mediated by the modulation of ADP pools by the light-dependent reactions of photosynthesis and ATP synthase because (1) two inhibitors that block ATP synthesis also prevent this activation and (2) ADP inhibits the RNA-binding activity of this protein in vitro. An inhibitor of Photosystem II diminishes this induction, suggesting that reducing potential generated by the photosynthetic electron transport chain modulates this RNA-binding activity. The RNA-binding activities of two proteins (of 46 and 47 kDa) are inhibited by Mg-protoporphyrin IX methyl ester in vitro suggesting they could be regulated by these intermediates in the chlorophyll biosynthetic pathway.     

Identification of a structure on human melanoma cells recognized by CTL exhibiting anomalous killer cell function

The structures involved in the recognition of melanoma cells by nonspecific cytotoxic T lymphocytes (CTL) activated in mixed lymphocyte culture were investigated with monoclonal antibodies (MAb) which blocked this anomalous killer (AK) function. Of over 2000 MAb raised against melanoma cells, only three inhibited killing; one of these, an IgMk termed Leo Me13, was investigated in detail. In antibody-binding studies using a large range of cultured tumor cells, it was shown that Leo Me13 was relatively specific for melanoma cells. Of more importance, Leo Me13 inhibited conjugate formation between AK cells and melanoma target cells by 60 to 80% and caused an eight- to 10-fold reduction in killing. The MAb did not immunoprecipitate protein from melanoma cells surface-labeled with 125I, and thin-layer chromatography followed by immunoblotting of the separated glycolipids from melanoma cells indicated that the epitope was on acidic glycolipids migrating between GM1 and GD1a; moreover, treatment of melanoma cells with neuraminidase resulted in complete loss of binding of Leo Me13 but not of other anti-melanoma antibodies which did not inhibit AK cell-mediated lysis. Other melanoma-reactive MAb of the same isotype as Leo Me13 did not block killing of melanoma cells, but one documented antibody, R24, an IgG3 with specificity for the ganglioside GD3, was found to inhibit this function. These data suggest that the AK cells recognize and bind to melanoma cells by a secondary "lectin-type" receptor for a carbohydrate moiety.     

Galactomannan and Zymosan Block the Epinephrine-Induced Particle Transport in Tracheal Epithelium

Background

Ciliary beating by respiratory epithelial cells continuously purges pathogens from the lower airways. Here we investigated the effect of the fungal cell wall polysaccharides Galactomannan (GM) and Zymosan (Zym) on the adrenergic activated particle transport velocity (PTV) of tracheal epithelium.

Methods

Experiments were performed using tracheae isolated from male C57BL/6J mice. Transport velocity of the cilia bearing epithelial cells was measured by analysing recorded image sequences. Generation of reactive oxygen species (ROS) were determined using Amplex Red reagents. PCR experiments were performed on isolated tracheal epithelium to identify adrenergic receptor mRNA.

Results

The adrenergic receptors α1D, α2A, β1 and β2 have been identified in isolated tracheal epithelium. We found epinephrine responsible for an increase in PTV, which could only be reduced by selective β-receptor-inhibition. In addition, either GM or Zym prevented the epinephrine induced PTV increase. Furthermore, we observed a strong ROS generation evoked by GM or Zym. However, epinephrine induced increase in PTV recovered in the presence of GM and Zym after application of ROS scavengers.

Conclusion

Both GM or Zym trigger reversible ROS generation in tracheal tissue leading to inhibition of the β-adrenergic increase in PTV.     

Analysis of the mechanisms of T cell-dependent polyclonal activation of human B cells. Induction of human B cell responses by fixed activated T cells.

Coculture of resting human B cells with T cells stimulated with immobilized mAb to the CD3 molecular complex induces polyclonal activation and the production of Ig of all isotypes. The current experiments were carried out to determine the nature of the signals provided to B cells by the anti-CD3-activated T cells. For these experiments, fresh T cells or T cell clones were activated with immobilized mAb to CD3 and then fixed with 1% paraformaldehyde. Upon coculture, the fixed activated T cells or T cell clones induced B cell RNA synthesis and IL-2R expression, but only minimal DNA synthesis and no Ig production. Induction of B cell RNA synthesis by fixed activated T cells was not inhibited by mAb to the alpha-chain of the IL-2R, and was not enhanced by supplementing cultures with IL-2, IL-4, IL-6, or supernatants of mitogen-activated T cells. Upon the addition of IL-2, but not IL-4 or IL-6, to cultures of B cells and fixed activated T cells, sustained proliferation was noted along with the production of Ig. Control fixed T cells or T cell clones did not induce any of these responses. The presence of cycloheximide or cyclosporin A during the activation with anti-CD3 prevented T cells from developing the capacity to provide help for B cells. The use of mAb to a variety of cell surface molecules indicated that several T cell surface molecules including CD11a/CD18, CD44, CD54, and class I MHC molecules are involved in the induction of B cell responses. Among the mAb that inhibited B cell DNA synthesis and/or Ig production, only mAb to CD11a, CD18, or CD54 inhibited initial B cell activation as assessed by RNA synthesis. Even though mAB to CD11a/CD18 inhibited the capacity of fixed activated T cells to induce B cell responses, the finding that fixed activated CD18 deficit clones provided help for B cells indicated that expression of the beta 2 family of integrins by T cells was not necessary. These results indicate that activated T cells acquire the capacity to stimulate B cells polyclonally and induce cytokine responsiveness, proliferation, and Ig production by utilization of a variety of surface molecules. Moreover, these results indicate that the initial activation of the B cell is independent of the metabolic activity of the T cell and the production of cytokines.