Modular Total Syntheses of the Alkaloids Discoipyrroles A and B, Potent Inhibitors of the DDR2 Signaling Pathway.

The title natural product 1 has been synthesized by treating the 1,2,3,5-tetrasubstituted pyrrole 23 with oxoperoxymolybdenum(pyridine) (hexamethylphosphoric triamide) (MoOPH). Compound 23 was itself prepared in seven steps from parent pyrrole using Ullmann-Goldberg and Suzuki-Miyaura cross-coupling, Vilsmeier-Haack formylation, electrophilic bromination, and Wittig olefination reactions as key steps. Related chemistry has been used to prepare discoipyrrole B (2).

Evaluación de la respuesta inmunológica del Apis Mellifica homeopatizada en cultivos de células mononucleares en sangre periférica / Evaluation of the immune response of the homeopathized Apis Mellifica in cultures of mononuclear cells in peripheral blood

Introducción: La Apis Mellifica, uno de los medicamentos homeopáticos utilizado por la homeopatía que tiene efectos sobre al sistema inmune, por ejemplo: actúa sobre la basófilos, provocando una inhibición en la liberación de histamina. El objetivo de este estudio es evaluar la respuesta de las células mononucleares frente al Apis Mellifica homeopatizada. Materiales y Métodos: La medición de los niveles de las citoquinas IL-1ß, IL- 2, IL-4, IL-6, IL-8 e IL-10, se determinaron cuando las células mononucleares en sangre periférica humana se expusieron al Apis Mellifica homeopatizada a la potencia 30 CH, mediante el ensayo "Cytokine Human Panel" de Invitrogen®. Resultados: Las células mononucleares crecieron un 18% en comparación con el grupo control, mostrando efectos mitogénicos del Apis Mellifica homeopatizada sobre las células mononucleares. Los resultados de los niveles de citoquinas IL-1ß,IL- 2, IL-4, IL-6, IL-8 e IL-10, medidos en el equipo de Luminex®, no evidencio modificación estadísticamente significativa de las mismas por parte del Apis Mellifica homeopatizada. Conclusiones: Los resultados del estudio mostraron un efecto estimulante de proliferación de las células mononucleares expuestas al Apis Mellifica homeopatizada, este estudio, deja la puerta abierta, para la realización de nuevos estudios.

Inhibition of collagen-induced discoidin domain receptor 1 and 2 activation by imatinib, nilotinib and dasatinib.

Imatinib, nilotinib and dasatinib are protein kinase inhibitors which target the tyrosine kinase activity of the Breakpoint Cluster Region-Abelson kinase (BCR-ABL) and are used to treat chronic myelogenous leukemia. Recently, using a chemical proteomics approach another tyrosine kinase, the collagen receptor Discoidin Domain Receptor1 (DDR1) has also been identified as a potential target of these compounds. To further investigate the interaction of imatinib, nilotinib and dasatinib with DDR1 kinase we cloned and expressed human DDR1 and developed biochemical and cellular functional assays to assess their activity against DDR1 and the related receptor tyrosine kinase Discoidin Domain Receptor2 (DDR2). Our studies demonstrate that all 3 compounds are potent inhibitors of the kinase activity of both DDR1 and DDR2. In order to investigate the question of selectivity among DDR1, DDR2 and other tyrosine kinases we have aligned DDR1 and DDR2 protein sequences to other closely related members of the receptor tyrosine kinase family such as Muscle Specific Kinase (MUSK), insulin receptor (INSR), Abelson kinase (c-ABL), and the stem cell factor receptor (c-KIT) and have built homology models for the DDR1 and DDR2 kinase domains. In spite of high similarity among these kinases we show that there are differences within the ATP-phosphate binding loop (P-loop), which could be exploited to obtain kinase selective compounds. Furthermore, the potent DDR1 and DDR2 inhibitory activity of imatinib, nilotinib and dasatinib may have therapeutic implications in a number of inflammatory, fibrotic and neoplastic diseases.

Discoidin domain receptor 1 mediates collagen-induced inflammatory activation of microglia in culture.

Discoidin domain receptor 1 (DDR1) is a nonintegrin collagen receptor tyrosine kinase with an extracellular domain homologous to discoidin 1 of a soil-living amoeba Dictyostelium discoideum. We have previously demonstrated that DDR1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages. Because collagen is one of the main components of extracellular matrix in the central nervous system, we hypothesized that collagen also induces inflammatory activation of brain microglia, and DDR1 may mediate collagen-induced microglial activation. Using BV-2 mouse microglial cells and mouse primary microglial cultures, we have demonstrated that (1) collagen induces inflammatory activation of microglia as evidenced by production of nitric oxide, expression of inducible nitric oxide synthase, COX-2, CD40, and matrix metalloproteinase-9; (2) DDR1 is expressed in microglia and is phosphorylated by collagen treatment; and (3) collagen-induced microglial activation is abrogated by DDR1 blockade but not by integrin neutralization. We have further shown that p38 MAPK, c-Jun N-terminal kinase, and nuclear factor-kappa B are involved in the collagen-DDR1-induced microglial activation. Our results suggest that collagen can induce inflammatory activation of brain microglia and that DDR1 mediates this effect of collagen in an integrin-independent manner.

Opposite effects on human colon cancer cell proliferation of two dietary Thomsen-Friedenreich antigen-binding lectins.

Increased cell surface expression of the Thomsen-Friedenreich antigen (TF antigen, Galbeta1-3GalNAcalpha-) is a common feature in malignant and pre-malignant epithelia. Our previous studies have shown that dietary TF-binding lectins from peanut (Arachis hypogea) and edible mushroom (Agaricus bisporus) produce marked but different effects on human intestinal epithelial cell proliferation. This study investigates the proliferative effects of the other two known dietary TF-binding lectins: jacalin (Artocarpus integrifolia, JAC) and amaranth lectin (Amaranthus caudatus, ACA). JAC produced dose-dependent and non-cytotoxic inhibition of proliferation in HT29 human colon cancer cells with maximal effects of 46 +/- 4% at 20 microg/ml, whereas ACA produced dose-dependent stimulation of proliferation with maximal effects of 22 +/- 3% at 20 microg/ml when assessed both by incorporation of [3H]thymidine into DNA and by cell counting. The lectin-mediated effects were inhibitable by the presence of appropriate Galbeta1-3GalNAc-expressing glycoproteins but differences existed between JAC and ACA in their patterns of inhibition by such substances. Ligand binding equilibrium studies using iodinated lectins revealed different Kd of the two lectins for HT29 cell surface glycoproteins. Lectin blots of cell membrane extracts showed different binding patterns in all the four TF-binding lectins. These results provide further evidence that dietary TF-binding lectins can have marked effects on the proliferation of human malignant gastro-intestinal epithelial cells and hence may play a role in intestinal cancer development, and also show that the biological effects of dietary lectins cannot be predicted solely from their carbohydrate binding properties.

Hydrogen peroxide inhibits epidermal growth factor receptor internalization in human fibroblasts.

Several cellular signal transduction cascades are affected by oxidative stress. In this study, the effect of hydrogen peroxide (H2O2) on the endocytosis of the epidermal growth factor (EGF) receptor was investigated. Exposure of HER14 cells to H2O2 resulted in a concentration-dependent inhibition of EGF receptor internalization. Binding studies demonstrated that this H2O2-induced inhibition in internalization was not due to altered binding of EGF to its receptor. Addition of H2O2 at different time points during internalization showed that EGF receptor internalization was rapidly reduced, suggesting that one of the first steps in the internalization process is inhibited. In addition, H2O2 inhibited the internalization of a different receptor, the chicken hepatic lectin receptor, in a concentration-dependent manner as well. Treatment of cells with another inducer of oxidative stress, cumene hydroperoxide, also resulted in a decreased internalization. Finally, we showed that H2O2 inhibited EGF-induced mono-ubiquitination of the EGF receptor pathway substrate clone 15, a process that normally occurs during EGF receptor endocytosis. These results clearly show that oxidative stress interferes with EGF signaling.

Mannan decelerates the clearance of human red blood cells in SCID mouse.

Mannans and its related compounds decelerated human (Hu) red blood cell (RBC)-clearance in severe combined immunodeficiency (SCID) mice by inhibiting erythro-phagocytosis of macrophages. Chimeric SCID mice for Hu-RBC which are generated by repeated transfusions with mature Hu-RBCs are described recently as a model for Plasmodium falciparum infection, though the Hu-RBC clearance in the mice at present is very rapid and the parasitemia in the mice is only erratic. Here, we aimed to study the method to decelerate Hu-RBC clearance in SCID mice, to establish a suitable mouse model for malaria parasites. Yeast and Candida mannans as well as lactoferrin, a glycoprotein containing both oligomannoside- and N-acetyllactosamine-type glycans, decelerated Hu-RBC clearance, but instead other saccharides such as carboxymethyl chitin, N-acetylglucosamine, and D-glucose did not. Yeast mannan and lactoferrin interfered significantly with in vitro Hu-RBC-phagocytosis which was also inhibited by mannopentaose and mannotoriose. D-mannose exhibited a moderate inhibitory activity. N-acetyl-D-glucosamine, however, showed only a slight inhibitory activity, but D-glucose had no inhibitory activity on Hu-RBC phagocytosis. These results may postulate that Hu-RBC clearance in SCID mouse might be mediated by receptor-ligand binding by a macrophage lectin like receptor with mannose specificity.

[The effect of nocodazole on the redistribution of phosphoinositide-specific phospholipase C gamma 1 during mitogenic signal transduction in A-431 cells]. / Vliianie nokodazola na pereraspredelenie fosfoinozitid-spetsificheskooi fosfalipazy C gamma 1 v protsesse peredachi mitogennogo signala v kletkakh A-431.

Phosphoinositide-specific phospholipase C gamma 1 is associated with EGF receptor in A-431 cells after EGF treatment. It is shown that phospholipase C gamma 1 is co-localized with internalized receptors as well as with membrane ones during receptor-mediated endocytosis. Nocodazole is known as an inhibitor of microtubule assembly, thus leading to a complete disappearance of microtubule network. Nocodazole had no effect on phospholipase C gamma 1--EGF receptor association and co-localization, but the intracellular distribution of both the proteins differed dramatically. Phospholipase C gamma 1 and EGF receptor were localized in endosomes in the periphery of the cell. Besides, pretreatment of A-431 cells with nocodazole resulted in decreasing tyrosine phosphorylation of some proteins. These data suggest that the internalized receptor may serve as an additional starting point for triggering cell signalling.

A study of effects of WGA and ConA on RBC membrane receptors using a new ektacytometric method.

With a new ektacytometry, we studied the relation between the microstructure of red blood cell (RBC) membrane and the rheological properties of RBCs in a shear flow field of low viscosity. The main contributions of this paper are as follows: 1. The hemorheological meanings of the orientation index (DI)or and the small deformation index (DI)d were explored. (DI)or is an overall rheological index depending on the deformability and morphology of RBCs. The better the physiological shape of RBCs is maintained, the greater the (DI)or is. (DI)d can be used to describe the lipid fluidity of RBC membrane. Such an explanation for the meaning of (DI)d has been forcefully supported by our experiments using electron spin resonance (ESR) and fluorescence polarization. 2. The influence of wheat germ agglutinin (WGA) of different concentrations on the lipid fluidity of membrane is different from that of concanavalin A (ConA). The lipid fluidity of membrane changes with WGA concentration treating RBCs and there is a maximum value for the membrane fluidity at a specific concentration of WGA. However, the deformability of membrane described by the integrate deformation index (IDI) monotonically decreased with the increase in WGA concentration treating RBCs. 3. It is concluded that the increase in the lipid fluidity of red cell membrane is not necessarily associated with the improvement of RBC deformability.

Lectin-like receptor for alpha 1-acid glycoprotein in the epithelium of the rat prostate gland and seminal vesicles.

BACKGROUND: A receptor for alpha 1-acid glycoprotein glycoforms AGP-B and AGP-C in the epithelium of the rat prostate gland and seminal vesicles is described. METHODS: The interaction between AGP-glycoforms and their receptor is a lectin-like interaction confirmed by inhibition of the binding by mannose and N-Acetyl-D-glucosamine. RESULTS: In vitro the receptor was also inhibited by the steroid hormones cortisone, aldosterone, progesterone, and estradiol, but not by testosterone. A significant regional variation in the expression of AGP-lectin receptor and in the localization of AGP was seen in rat prostate and seminal vesicles. The localization of the AGP lectin receptor is compared to the localization of glycoprotein AGP, and small differences are found. CONCLUSIONS: It is proposed the AGP receptors in the prostate and seminal vesicles belong to a group of lectins in the control of differentiation and organ formation.

Influence of culture media and multidrug resistance on the wheat germ agglutinin (WGA) glycocytochemical expression of two human glioblastoma cell lines.

Over the last two decades, many studies carried out with the aid of lectins have firmly established that cell glycans usually change in the course of the normal processes of growth and development, as well as in pathological situations. We describe here the in vivo binding expression of wheat germ agglutinin (WGA) to the U87 and U373 human glioblastoma cell lines exposed to various culture media i.e., media supplemented with either 10% (FCS10) or 1% (FCS1) fetal calf serum with or without 10 n Mol/l 17 beta-oestradiol (E2). After exposure to chemotherapeutic agents, the resistant variants (CR) developed by the two cell lines were also investigated. The quantitative cytochemical assessment of WGA binding was assessed by means of a cell image processor, which was also used to determine ploidy level (on Feulgen-stained nuclei) by means of DNA histogram typing (DHT). Our results clearly demonstrate that when U373 cells are cultured with E2, this steroid can modify the expression of WGA binding, whereas U87 cells were unaffected. Similarly, lowering the FCS level enhanced the WGA binding of the U373 cell line. Multidrug-resistant cell variants were associated with both aneuploidy and a dramatic decrease in cytochemical WGA expression.

Enhancement of phagocytosis by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages.

Calcitonin gene-related peptide (CGRP) is widely distributed in sensory neurons and nerve fibers. To clarify the function of CGRP on the immune system, the effect of CGRP on phagocytosis by peritoneal macrophages was examined by means of flow cytofluorometry. CGRP enhanced phagocytosis of latex beads in a dose-dependent manner. Because the phosphodiesterase inhibitor 3-isobutyl, 1-methylxanthine (IBMX) enhanced the CGRP-induced enhancement of phagocytosis, the enhancement might be mediated by cAMP. In the presence of mannan, the phagocytosis was suppressed and the CGRP-induced enhancement was also blocked, suggesting that mannose receptors on macrophages were involved in mediating the phagocytosis of latex beads, and CGRP enhanced the mannose receptor-mediated phagocytosis. The present results indicate that CGRP can modulate the function of macrophages in nerve terminals of sensory neurons during the development and maintenance of inflammation.

Vascular endothelial growth factor and its receptors.

Vascular endothelial growth factor (VEGF) is a highly specific mitogen for vascular endothelial cells and an angiogenic factor that is structurally related to platelet derived growth factor (PDGF). It is also known as the vascular permeability factor (VPF) because it efficiently potentiates the permeabilization of blood vessels. Five types of VEGF mRNA encoding VEGF species which differ in their molecular mass and in their biological properties are transcribed from a single gene as a result of alternative splicing. VEGFs are produced and secreted by several normal cell types including smooth muscle, luteal and adrenal cortex cells. VEGFs are also produced by different tumorigenic cells, and appear to play a major role in tumour angiogenesis. Antibodies directed against VEGF can inhibit the growth of a variety of VEGF producing tumours. Of the various VEGF species, the best characterized is the 165 amino acid long form (VEGF165). VEGF165 is a heparin binding growth factor, and its interaction with VEGF receptors on the cell surface of vascular endothelial cells depends on the presence of heparin-like molecules. Several cell types which do not proliferate in response to VEGF such as bovine corneal endothelial cells, HeLa cells and human melanoma cells also express cell surface VEGF receptors, but the function of the VEGF receptors in these cells is unclear. Recently, the tyrosine-kinase receptors encoded by the flt and KDR/flk-1 genes were found to function as VEGF165 receptors.

Characterization of interleukin-4 receptors expressed on human renal cell carcinoma cells.

We have recently reported that a variety of solid human tumor cells express high-affinity interleukin-4 receptors (IL-4R). In this study, we have compared structural characteristics of IL-4R expressed on human renal cell carcinoma cells (RCC-WS) and the lymphoid cell lines RAJI (B-cell line) and H9 (T-cell line). In crosslinking studies, the three cell types expressed a predominant 140 kDa IL-4R band. In addition, a 70 kDa band was expressed strongly in H9 cells but only faintly on RCC-WS and RAJI cells. These different species of IL-4R were not observed when crosslinking studies were performed in the presence of excess interleukin-4 (IL-4), indicating IL-4 specificity. A polyclonal anti-IL-4R antibody immunoprecipitated the two species (140 and 70 kDa) in H9 and predominantly the 140 kDa species in RCC-WS tumor cells and RAJI cells. A faint band for the 70 kDa protein was also observed. The affinity of IL-4 binding to its receptor in RCC-WS cells was similar to the binding affinity observed in H9 and RAJI cells examined. However, the RCC tumor cells and B lymphoid cells internalized IL-4R more rapidly compared to T lymphoid cells. Although IL-4R synthesis was similarly inhibited by cycloheximide in all three cell lines, IL-4R expression was more sensitive to actinomycin D inhibition on the RCC-WS and RAJI cells than on H9 cells. Our results suggest that IL-4R expressed on RCC-WS tumor cells are structurally different from those expressed on lymphoid cells because the proportions of IL-4R subunits differ in these cells. Further studies should be performed to determine the identity and functional significance of IL-4R proteins expressed on RCC and immune cells.

Studies on the effect of mevinolin (lovastatin) and mevastatin (compactin) on the fusion of L6 myoblasts.

The effect of mevastatin and mevinolin on the fusion of L6 myoblasts was studied. Both compounds were present inhibitors of myoblast fusion at concentrations as low as 0.25 microM, but fusion was restored when the inhibitors were removed. Both compounds resulted in decreased binding of conA and WGA to cell surface oligosaccharides showing they were causing a reduction in N-linked cell surface glycoproteins. There was a reduction in creatine phosphokinase activities in the presence of both compounds showing that they were affecting biochemical differentiation. The presence of both compounds inhibited the incorporation of labeled mannose from GDP-mannose into lipid-sugar and N-linked glycoprotein, but the inhibition was reversed by addition of exogenous dolichol phosphate to the incorporation mixture. The main conclusion from these studies is that mevinolin and mevastatin are inhibiting myoblast fusion by affecting the synthesis of fusogenic cell surface N-linked glycoproteins probably by affecting the synthesis of dolichol phosphate containing oligosaccharides that are required as intermediates in N-linked glycoprotein biosynthesis.

A wide range of human cancers express interleukin 4 (IL4) receptors that can be targeted with chimeric toxin composed of IL4 and Pseudomonas exotoxin.

A chimeric toxin has been constructed by fusion of a gene encoding human interleukin 4 (hIL4) to a gene encoding a mutant form of Pseudomonas exotoxin A (PE) which cannot bind to its receptors (PE4E). The chimeric gene was expressed in Escherichia coli where large amounts of the chimeric toxin, hIL4-PE4E, was produced. Purified hIL4-PE4E was very cytotoxic to cancer cell lines of both hematopoietic and solid tumor origin. In the HUT 102 T cell leukemia and Daudi B cell lymphoma cell lines, protein synthesis was inhibited by 50% (ID50) at a hIL4-PE4E concentration of 2 and 7 ng/ml (25 and 86 pM, respectively). hIL4-PE4E was also very cytotoxic to cell lines derived from carcinomas of the colon, breast, stomach, liver, adrenals, and prostate, as well as melanoma and epidermoid carcinoma, indicating that hIL4 receptors are widely expressed on human malignancies. We also found that human phytohemagglutinin-activated peripheral blood lymphocytes were extremely sensitive to hIL4-PE4E with an ID50 of 0.2 ng/ml (2.5 pM). The cytotoxic action of hIL4-PE4E was specific because it was blocked by an excess of hIL4 and not of human interleukin 2. In addition, hIL4-PE4ED553, an enzymatically inactive form of the chimeric toxin, was not cytotoxic. These results suggest that the hIL4 receptor may be a target for therapy in malignant and immunologic disorders using hIL4 chimeric toxin.

Expression of high affinity interleukin-4 receptors on human renal cell carcinoma cells and inhibition of tumor cell growth in vitro by interleukin-4.

Previously, Puri et al. (Puri, R. K., M. Ogata, P. Leland, G. M. Feldman, D. Fitzgerald, and I. Pastan. 1991. Cancer Res. 51:3011-3017) have demonstrated that murine sarcoma and colon adenocarcinoma cells express high affinity interleukin-4 receptors (IL-4R) which are internalized after binding to a chimeric ligand consisting of IL-4 and Pseudomonas exotoxin. In the present study, we have tested primary cultures of human renal cell carcinoma (RCC) cells, generated from tumor specimens obtained after nephrectomy, for the expression of IL-4R and their modulation by IL-4. By using iodinated IL-4 in a receptor binding assay, we observed that renal cell carcinoma cells expressed a single class of high affinity IL-4R ranging from 1,425 +/- 207 (mean +/- SEM) to 3,831 +/- 299 (mean +/- SEM) IL-4R molecules/cell with a Kd ranging from 112 +/- 11 pM to 283 +/- 71 pM. Northern blot analysis for IL-4R gene expression, performed with a cDNA probe to IL-4R, revealed that all RCC cells exhibited a single mRNA species of 4 kb. IL-4 downregulated the surface expression of IL-4R on one RCC tumor cell line. The function of IL-4R expression on RCC tumor cells was further determined by investigating the effect of IL-4 on tumor cell growth in vitro and comparing it with IL-4 effect on growth of normal fibroblast and endothelial cell lines. Tumor cell growth, as measured by [3H]thymidine incorporation, was inhibited by IL-4 from 20 to 68% in a dose-dependent manner. A neutralizing antibody to human IL-4 was able to reverse the growth inhibitory effect of IL-4. Normal human fibroblast and endothelial cell lines also expressed high affinity IL-4R, however, IL-4 did not inhibit their growth in vitro. In fact, IL-4 caused modest stimulation of their growth. Taken together, our findings can help develop strategies for the treatment of RCC in which IL-4R may be used as a target for IL-4 itself, for IL-4 toxin therapy or, alternatively, in gene therapy.

Growth factors accelerate epithelial repair in sheep trachea.

Toxic gases and fumes have been shown to be injurious to the upper airways. Repair of this injury involves proliferation and migration of surviving nonciliated cells, followed by differentiation to a normal phenotype. Because recent results suggested that growth factors could improve the outcome of an airway injury, we undertook this study to determine the efficacy of these materials as an initial treatment to accelerate the healing process. In 24 anesthetized sheep, a portion of the trachea was exposed to smoke from smouldering cotton cooled to 37 degrees C. Twelve received aerosolized epidermal growth factor plus platelet derived growth factor, while twelve received placebo. At 10 days after injury, nonciliated and ciliated cells were totally absent in the injured trachea receiving the placebo. In animals receiving growth factors, nonciliated and ciliated cells, however, were present (56% and 31% of uninjured trachea, respectively). At 13 days after injury, nonciliated and ciliated cell counts in those receiving placebo were 67% and 33% of uninjured, respectively. In sheep receiving growth factors, tracheal nonciliated and ciliated cell counts had increased to 105% and 64% of uninjured trachea, respectively. We conclude that growth factors therapy after airway injury stimulates cell proliferation and differentiation, and this therapeutic intervention to accelerate the repair process in acute airway injury is an approach applicable to humans.

A putative lectin-binding receptor mediates cadmium-evoked calcium release.

Nanomolar concentrations of cadmium (Cd2+) produce an immediate rise in free Ca2+ in human dermal fibroblasts, which is mostly caused by the release of stored Ca2+ via inositol trisphosphate. Here we have used lectins to evaluate the hypothesis that a cell surface glycoprotein mediates the response to Cd2+. A prior incubation with wheat germ agglutinin (WGA) or certain other lectins inhibited calcium release evoked by Cd2+. WGA reversibly inhibited Cd(2+)-evoked calcium release as indicated by measurements of cytosolic free Ca2+ and 45Ca2+ efflux. WGA half-maximally inhibited Ca2+ release at 1.2 x 10(-7) M. The Kd for the binding of fluoresceinylated WGA was 2.8 x 10(-7) M. Chitotriose dissociated fluoresceinylated WGA from the cells and restored cadmium responsiveness. WGA inhibited Cd(2+)-evoked 45Ca2+ efflux similarly at 18 and 37 degrees C. A brief incubation with chitotriose at 18 or 10 degrees C reversed the inhibition by WGA. WGA neither bound 109Cd2+ nor affected 109Cd2+ uptake by the cells. Succinylated WGA, which binds N-acetylglucosamine but not N-acetylneuraminic acid, failed to inhibit Ca2+ release evoked by Cd2+. WGA probably inhibits Ca2+ release produced by Cd2+ by binding to N-acetylneuraminic acid in the external domain of a plasma membrane receptor.

Identification of an essential region for growth signal transduction in the cytoplasmic domain of the human interleukin-4 receptor.

Interleukin-4 (IL-4) is a pleiotropic lymphokine which plays an important role in the immune system by regulating proliferation and differentiation of a wide variety of lymphoid and myeloid cells. These biological effects are manifested via binding of IL-4 to specific membrane-associated high affinity receptors. While the IL-4 receptor (IL-4R) cDNA expresses high affinity binding sites when transfected in COS7 cells, its intracellular domain lacks consensus motifs for known signal transducing molecules such as a tyrosine kinase. In this study, we use a DNA deletion approach to explore the mechanism of signal transduction utilized by the human IL-4R cDNA expressed in a murine pro-B cell line, Ba/F3 cells. Using this system, we have identified the critical region of the cytoplasmic domain of human IL-4R for human IL-4-induced transduction of a growth signal in these cells. Our data indicate that the critical region for signal transduction is located between amino acid residues 433-473 numbering from the carboxyl terminus. This region is highly conserved between mouse and human IL-4R but lacks homology with other cytokine receptors. Our studies additionally demonstrate that the cytoplasmic domain is not essential for forming high affinity IL-4-binding sites nor for ligand internalization.