Translocation of glucokinase in pancreatic beta-cells during acute and chronic hyperglycemia

We recently reported that Fc mu R on NK cells is a signal transducing protein that stimulates a rapid increase in the level of cytoplasmic free calcium upon binding of IgM. This study was designed to examine signal transduction via the Fc mu R on NK cells and to characterize intracellular second messengers activated by IgM. Immunoprecipitation of IgM-bound Fc mu R by IgM-specific Ab coimmunoprecipitated the zeta- and Fc epsilon RI gamma-chains. Furthermore, engagement and clustering of Fc mu R by polyclonal IgM induced tyrosine phosphorylation of the zeta- and Fc epsilon RI gamma-chains, indicating their functional association with the Fc mu R-induced signal transduction cascade. Ligand-induced clustering of the Fc mu R also induced activity of src family kinases, Lck, Fyn, Lyn, and Src, as well as their physical interaction with the receptor. Triggering via Fc mu R also induced the activity of Syk and Zap-70, tyrosine kinases demonstrated to associate with zeta and Lck. Phospholipase C-gamma 1 and phosphatidylinositol 3-kinase were identified as substrates phosphorylated on tyrosine, as down-stream components of the signaling pathway activated in NK cells by polyclonal IgM. Although the Fc mu R on NK cells has not yet been biochemically characterized, our results suggest that the zeta- and Fc epsilon RI gamma-chains are functional subunits of this as well as other important cell surface receptors and that the Fc mu R is coupled either directly or indirectly to nonreceptor tyrosine kinases, which phosphorylate and thereby activate regulatory enzymes such as phospholipase C-gamma 1 and phosphatidylinositol 3-kinase.     

Brownian dynamics study of ion transport in the vestibule of membrane channels

The Immunoglobulin (Ig) binding capacity of Toxoplasma gondii tachyzoites was investigated using fluorescence flow-cytometry analysis. Polyclonal mouse, human and rat immunoglobulins without specific anti-Toxoplasma activity bound to parasites in a concentration-dependent manner, saturating them at circulating serum concentrations. The immunoglobulin class and subclass specificity of binding was investigated using irrelevant monoclonal antibodies. IgM, IgA and IgG reacted with the parasite membrane. The attachment of mouse IgM to the parasite surface was hampered by mouse IgG1, IgG2a, IgG2b and IgG3. The binding of mouse IgG was proportionally reduced with increasing concentrations of mouse monoclonal IgM. The binding of murine immunoglobulin was diminished when in presence of human IgG. Purified Fc- but not Fab portions of immunoglobulins, fixed to parasites. Using labelled calibrated beads, the Ig binding capacity of parasites was estimated to be 6900 +/- 500 sites per tachyzoite. The Kd of the T. gondii Fc Receptor (FcR) activity was determined at 1.4 +/- 0.1 microM (mean +/- SEM). Such FcR activity was reduced by phospholipase C, trypsin and pronase treatment of the parasites. These data show a low affinity FcR activity on T. gondii tachyzoites which recognizes Ig of different species and isotypes and is likely supported by a glycosyl-phosphatidylinositol (GPI)-anchored surface protein of the parasite.     

Surface makers for mast cell subtypes: low affinity IgG receptors and gp49 family

Members of the Immunoglobulin Superfamily (Ig) present in the surface of rodent mast cells include the high affinity IgE receptor (Fc epsilon RI), the low affinity receptors for the Fc portion of IgG, the Fc gamma RII family and Fc gamma RIII as well as the recently cloned gp49 family that includes three members gp49A, gp49B1 and gp49B2. Fc epsilon RI and Fc gamma RIII are members of the multi-chain immune recognition receptor (MIRR) family since they possess a multimeric structure in which the signal transducing chains (gamma chains) contain six acids that conform the Antigen Homology Receptor 1 Motif (ARH1M), also present in the T cell receptor (TCR) transducing chains. gp49B1, gp49B2 and the FC gamma R family are monomeric chains that also contain the partial of full AHR1M motif in their cytoplasmic domain indicating the capability for signal transduction through tyrosine phosphorylation and the possibility of cell activation with mediator (s) or cytokine (s) release. Distribution of the Fc gamma R receptors and gp49 family members varies in the different stages of mast cell differentiation and maturation.     

B cell antigen receptor desensitization: disruption of receptor coupling to tyrosine kinase activation

Antigen binding to the B cell receptor (BCR) induces receptor desensitization, a condition characterized by cellular unresponsiveness to subsequent Ag stimulation despite the continued ability to bind Ag. To better understand the molecular mechanism of this unresponsiveness, we have used complementary lymphoma (K46 mu) and Ig transgenic (3-83 mu delta) mouse models to study regulation of BCR signaling. Our findings in the lymphoma model show that an initial Ag encounter renders receptors unresponsive to subsequent Ag challenge, as measured by their inability to mobilize Ca2+ and to mediate phosphorylation of receptor-proximal kinases, including Lyn, Blk, and Syk. Most importantly, the Ig alpha and Ig beta components of desensitized receptors are not phosphorylated, and receptor-associated kinases are not activated upon Ag challenge. The molecular defect does not appear to result from Lyn inactivation, sequestration, or repression, since Lyn from desensitized cell lysates is activated in vitro by synthetic doubly phosphorylated immunoreceptor tyrosine-based activation motif peptides. A similar deficit in Ag-induced receptor phosphorylation was observed in desensitized B cells from 3-83 mu delta transgenic mice. These studies indicate that Ag receptor desensitization reflects an inability to initiate activation of receptor-associated kinases that normally phosphorylate receptor Ig alphabeta subunits, leading to signal propagation.     

Muscle energy technique: definition, explanation, methods of procedure

The essential findings in this paper are that ox erythrocytes (ORBC) sensitized with human heterophile IgM antibody failed to detect Fc mu receptors on human lymphocytes. These results contrast to those obtained when the ORBC were sensitized with mouse hybridoma IgM antibody (SCC I). It is therefore suggested that the functional role of Fc mu receptors on human lymphocytes is not to bind IgM/antigen complexes.     

Negative signaling in B cells causes reduced Ras activity by reducing Shc-Grb2 interactions

To elucidate the molecular basis for inhibition of B cell proliferation and differentiation by the Fc receptor for IgG (Fc(gamma)RII), we compared the signaling events in B cells stimulated by cross-linking surface Ig alone (positive signaling), or by co-cross-linking surface Ig and Fc(gamma)RII (negative signaling). Both modes of stimulation induced tyrosine kinase activation. Positive signaling induced activation of Ras, Raf-1 kinase, and mitogen-activated protein kinase; these events were significantly attenuated during negative signaling. Since Ras is activated by SOS and Vav, two known guanine nucleotide exchange factors, activation events associated with these molecules using the two different stimuli were examined. Results of these experiments indicated that tyrosine phosphorylation of Vav did not change upon co-cross-linking. In contrast, the association of Shc and Grb2 was abrogated under negative and induced under positive signaling conditions. Concomitantly, Shc was observed to associate with a tyrosine-phosphorylated 145-kDa protein, previously identified as Src homology 2-containing inositol phosphatase, only under conditions of negative signaling. Based on these results, we hypothesize that negative signaling via the Fc(gamma)RII in B cells is at least partly the result of a block in Ras activation, and that SOS, but not Vav, is the major guanine nucleotide exchange factor in B cells for Ras activation.     

Role of mu heavy chain in B cell development. I. Blocked B cell maturation but complete allelic exclusion in the absence of Ig alpha/beta

There is good evidence for a signaling role played by Ig heavy chain in the developmental transition through the pre-B cell stage. We have previously described signal-capable or signal-incapable mutants of mu heavy chain in which a signaling defect is caused by failure to associate with the Ig alpha/beta heterodimer. To further characterize the role of Ig heavy chain-mediated signaling in vivo, as well as in B cell development and allelic exclusion, we have created transgenic mice in which the B cells express these signal-capable and signal-incapable mutant mu chains. Failure of mu to signal via Ig alpha/beta results in a block in B cell development in mice expressing the signal-incapable mu. A small number of B cells in these animals do escape the developmental block and are expressed in the spleen and the periphery as B220+ transgenic IgM+ cells. These cells respond to LPS by proliferating but show no response to T-independent-specific Ag. In contrast, B cells expressing the signal-capable B cell receptor show a strong signaling response to Ag-specific stimulus. There is no Ig alpha seen in association with signal-deficient IgM. Thus, the B cell receptor complex is not assembled, and no signal can be delivered. Despite the block in developmental signaling, allelic exclusion is complete. There is no detectable coexpression of transgenic IgM and endogenous murine IgM, nor is there rearrangement of the endogenous heavy chain genes. This suggests that differing signaling mechanisms are responsible for the developmental transition and allelic exclusion and thus allows for separate examination of these signaling mechanisms.     

The development of anti-CD79 monoclonal antibodies for treatment of B-cell neoplastic disease

The B-cell antigen receptor (BCR) consists of cell surface IgM associated with the CD79 alpha/beta heterodimer. In this paper we describe a panel of monoclonal antibodies (mAbs) recognising the extracellular regions of human CD79 alpha and beta. FACS analysis demonstrated that the mAbs bind to a range of Burkitt's lymphoma lines, a mouse B-cell line (JO-72) transfected with human CD79 alpha and beta, and tumour biopsies from NHL patients. The specificity of the mAbs was confirmed by immunoprecipitation. The Ka for the binding of IgG from the anti-CD79 alpha mAbs to cell surface CD79 alpha on Ramos cells was 3 x 10(8) M-1, and their maximum level of binding, 1.7-2 x 10(5) molecules/cell, matched that obtained with anti-Fc mu and anti-Fd mu mAbs. All four anti-CD79 beta mAbs were of lower affinity. Interestingly, in growth arrest studies, we found that while all anti-Fc mu mAbs caused profound inhibition of proliferation of Ramos cells, a range of other anti-BCR mAbs, which included the anti-CD79, anti-Fab mu, anti-gamma and anti-idiotype reagents, all performed poorly giving a maximum of 25% inhibition. These differences in performance are believed to relate to the ability of anti-BCR mAbs to cross-link neighbouring surface BCR and suggest that, unlike anti-Fc mu which favours cross-linking, most of these mAbs are binding in a monogamous, non-cross-linking, union with the BCR.     

Immunochemical properties of a 60 kDa cell surface-associated heat shock-protein (Hsp60) from Helicobacter pylori

Western blot analysis (immunoblotting) of cell surface-associated proteins from Helicobacter pylori confirmed our previous findings that binding of human IgG is a common property (among H. pylori strains). Purification of the IgG-binding proteins (IGBP) was achieved by two purification steps, affinity chromatography on IgG-Sepharose and nickel chelate affinity chromatography. SDS-PAGE and immunoblotting analysis revealed a 60 kDa protein with affinity for peroxidase labeled human IgG. Solid phase binding assays showed that IgG binds to an immobilized protein (IGBP). The 60 kDa IGBP binds human IgG1, IgG3 and IgM. Binding could be inhibited by the kappa chain of the human IgG, but not with its Fc fragment, nor with IgA or IgM. In addition, rabbit polyclonal antibodies raised against the 60 kDa IGBP blocked IgG binding. Monoclonal antibodies, specific to the Hsp60 heat shock protein of H. pylori recognized the 60 kDa IGBP as revealed by immunoblotting analysis, both in crude preparations and in the purified fractions.     

EA rosette-forming lymphoid cells in chickens: specificity of the Fc receptor and its relationship to other surface antigens

The receptor for erythrocyte-antibody (EA) complexes on the surface of chicken lymphoid cells was investigated using a rosette assay. The chicken EA receptor binds chicken immunoglobulin of the IgG class but not the IgM class. Binding to the EA receptor is dependent upon the Fc region of the immunoglobulin. No receptor for complement analogous to the mammalian C3b receptor was demonstrated on chicken lymphoid cells using the rosette assay. Inhibition studies utilizing immunoglobulins from several species demonstrated that chicken spleen cells do not bind mammalian immunoglobulin but may bind immunoglobulin of other avian species (turkey and duck) and a reptilian species (turtle). The chicken EA receptor is distinct from cell membrane bound immunoglobulin light chains, bursa-specific antigens and thymus-specific antigens. The receptor for EA complexes on chicken lymphoid cells is compared with the Fc receptor on mammalian lymphoid cells in the light of these observations.     

Structure and function of immunoglobulin domains. V. Binding, University of immunoglobulin G and fragments to placental membrane preparations

Fc receptors have been shown to be present in human placental tissue with properties distinct from those on macrophages and lymphocytes. A single class of receptor was observed with an intrinsic affinity 4X 10(6) M-(1) for human IgG1. The order of affinity for IgG subclasses was IgG1 = IGG1 greater than IgG3 greater than IgG4. IgA and IgM were not bound. Fc from IgG1 bound with the same affinity as the whole molecule and to the same number of receptor sites, 2 X 10 (12)/mg placental protein. IgG1 was no longer boung after mild reduction and alkylation whereas the binding of Fc was unaffected by this treatment. Neithe C3 nor C3, the two domains which comprise the Fc region of IgG1, bound to the placental receptor. This implies that this Fc receptor is unlike those found on most cell surfaces and that plasental binding is an exception to the theory that each domain has evoked to perform independent functions.     

Characterization of the human immunoglobulin G Fc-binding activity in Prevotella intermedia

Many pathogenic bacteria possess cell surface receptors which can bind immunoglobulins via the Fc portion. The aim of this study was to characterize the human immunoglobulin G (IgG) Fc-binding activity of Prevotella intermedia, a suspected etiologic agent of adult chronic periodontitis. The Fc-binding activity of P. intermedia on whole cells and on extracellular vesicles was demonstrated. Incubation of P. intermedia cells in the presence of Zwittergent 3-14 allowed complete solubilization of the Fc receptor from the cell surface. This cell envelope extract was thus used to characterize the Fc-binding activity. A microtiter plate assay using alkaline phosphatase-labeled Fc fragments showed that preincubation of the cell envelope extract with human IgG, human IgG Fc fragments, or human serum completely inhibited the Fc-binding activity. Partial inhibition was obtained with human IgG F(ab')2 fragments, whereas no inhibition occurred following preincubation with human IgA, carbohydrates, and selected proteins. Preincubation of the cell envelope extract with IgG from a variety of animals demonstrated that rabbit, mouse, rat, goat, and sheep IgG did not inhibit Fc-binding activity, whereas cow, pig, and dog IgG partially inhibited Fc-binding activity. A strong inhibition comparable to that obtained with human IgG was noted with monkey IgG. The Fc receptor of P. intermedia is thus different from the six types previously reported in other nonoral bacteria. Polyacrylamide gel electrophoresis and Western blotting (immunoblotting) analysis of the cell envelope extract revealed a major band with a molecular mass of approximately 65 kDa which reacted with peroxidase-labeled human IgG Fe fragments. Transmission electron microscopy showed a uniform distribution of the Fc receptor on the bacterial surface, as revealed by gold labeling. The Fc-binding activity demonstrated in this study may act as an additional virulence factor for P. intermedia by reducing IgG reactions with the bacterial cell.     

Structure and function of human IgA Fc receptors (Fc alpha R)

Receptors for the Fc region of IgA are expressed by many human cell types, especially phagocytes located in mucosal areas, where IgA is the prevalent antibody isotype. Binding of IgA-opsonized particles (e.g., bacteria, viruses) to Fc alpha R may trigger a plethora of cell-mediated immune effector functions designed to rid the body of the foreign invader. The IgA receptor present on myeloid cells such as neutrophils, eosinophils, and monocytes (Fc alpha RI or CD89) is a transmembrane glycoprotein that binds both IgA isotypes with similar affinity. Genetic characterization showed Fc alpha RI to be a more distantly related member of the Ig receptor gene family. Recently, Fc alpha RI was found to associate with the FcR gamma-chain signaling molecule through a unique charge-based mechanism. Fc alpha RI is, thus, connected to the intracellular machinery via the ITAM signaling motifs located within the cytoplasmic tail of FcR gamma-chain. Evidence exists in support of receptors for IgA (distinct from Fc alpha RI) on human T and B cells. IgA Fc receptors may, therefore, play a role in both the induction and control of an efficient (mucosal) immune response.     

Detection and biochemical characteristics of the receptor for complexes of soluble CD14 and bacterial lipopolysaccharide

Soluble CD14 (sCD14) has been found to bind LPS and mediate LPS activation of several cell types. It has been postulated that sCD14-LPS complexes induce cell responses by interacting with a cell surface structure, which, in turn, triggers cell activation. There has been no biochemical evidence, however, for a direct interaction of sCD14 with a cell surface structure, and the putative receptor has not been identified. To rigorously test this hypothesis, we studied the interaction of human rsCD14 with cells in the absence of serum and in the presence and the absence of LPS. We found 1) there was specific and saturable binding of 125I-sCD14, indicative of a typical receptor-ligand interaction, to several cell types, including endothelial cells, epithelial cells, astrocytes, and human monocytes; 2) specific binding to all the cell types and IL-6 induction in membrane-bound CD14 (mCD14)-negative cells occurred only when both sCD14 and LPS were present; 3) competitive displacement experiments of 125I-sCD14 binding to astrocytes and Scatchard plots revealed a binding of high affinity (Kd = 3.3 +/- 0.4 nM) and approximately 25,000 single class binding sites/cell; 4) the steady state for the association of 125I-sCD14 was obtained after 180-200 min; 5) chemical cross-linking experiments revealed the association of sCD14 with a binding structure of approximately 216 kDa; 6) binding of 125I-sCD14 to CD14-expressing cell transfectants was about 50% lower than that to nontransfected cells. Maximal binding, however, was recovered after removing mCD14, suggesting that the sCD14-LPS receptor may also interact with mCD14. These results provide direct biochemical evidence for the existence of a cell surface signal-mediating binding structure for LPS-bearing sCD14 and suggest that this structure may represent the signaling unit of the postulated multimeric LPS receptor in mCD14-bearing cells.     

Stabilization and activation of p53 are regulated independently by different phosphorylation events

Rheumatoid factors (RF) recognize conformational determinants located within the Fc portion of IgG. By analyzing a panel of monoclonal rheumatoid arthritis (RA)-derived RFs, we previously demonstrated that the somatically generated light chain complementarity-determining region 3 (CDR3) contributes to RF specificity. We have now generated a panel of heavy chain mutants of the B'20 Ab, a high affinity RA-derived IgM RF. B'20 also binds avidly to protein A and weakly to ssDNA and tetanus toxoid. B9601, a RF negative Ab that is highly homologous to B'20 but does not bind any of the Ags tested, and RC1, a low affinity polyreactive RF, were used to generate heavy chain mutants with framework (FR) and CDR switches. The mutated heavy chains were cotransfected into a myeloma cell line with the germline counterpart of the B'20 light chain, and the expressed Ig tested for antigenic specificity. We show that both RF specificity and polyreactivity of B'20 is dependent on its unique heavy chain CDR3 region. Replacement with a B9601 CDR3 shortened to the same length as the B'20 CDR3, and with only 5 amino acid differences, did not restore Fc binding. Conversely, absence of protein A binding of B9601 is due to the presence of a serine residue at position 82a in the B9601 heavy chain FR3 region. Together, our data suggest that Ig gene recombination events can generate B cells with autoantibody specificities in the preimmune repertoire. Abnormal release, activation, expansion, or mutation of such cells might all contribute to the generation of a high titer RF response in patients with RA.     

Role of the extracellular domain of human herpesvirus 7 glycoprotein B in virus binding to cell surface heparan sulfate proteoglycans

In an attempt to identify the human herpesvirus 7 (HHV-7) envelope protein(s) involved in cell surface binding, the extracellular domain of the HHV-7 glycoprotein B (gB) homolog protein was cloned and expressed as a fusion product with the Fc domain of human immunoglobulin G heavy chain gamma1 (gB-Fc) in an eukaryotic cell system. Indirect immunofluorescence followed by flow cytometric analysis revealed specific binding of gB-Fc to the membrane of SupT1 cells but not to other CD4+ T-lymphoblastoid cell lines, such as Jurkat or PM1, clearly indicating that gB-Fc did not bind to the CD4 molecule. This was also suggested by the ability of gB-Fc to bind to CD4-negative fibroblastoid Chinese hamster ovary (CHO) cells. The binding was abrogated by enzymatic removal of cell surface heparan sulfate proteoglycans by heparinase and heparitinase but not by treatment with condroitinase ABC. In addition, binding of the gB-Fc fusion protein to CHO cells was severely impaired in the presence of soluble heparin, as well as when heparan sulfate-deficient mutant CHO cells were used. Consistent with these findings, soluble heparin was found to block HHV-7 infection and syncytium formation in the SupT1 cell line. Although the CD4 antigen is a critical component of the receptor for the T-lymphotropic HHV-7, these findings suggest that heparin-like molecules also play an important role in HHV-7-cell surface interactions required for infection and that gB represents one of the HHV-7 envelope proteins involved in the adsorption of virus-to-cell surface proteoglycans.     

A tyrosine-based signal present in Ig alpha mediates B cell receptor constitutive internalization

B lymphocytes express Ag receptors (BCR) that are composed of ligand binding subunits, the membrane Igs, associated with Ig alpha/Ig beta heterodimers. One main BCR function is to bind and to internalize Ags. Peptides generated from these internalized Ags may be presented to T lymphocytes. Here, we have analyzed the involvement of BCR Ig alpha/Ig beta components in BCR constitutive endocytosis. The role of Ig alpha subunit in BCR constitutive endocytosis was first determined in the context of an IgM-based BCR. In contrast with BCR that contain wild-type Ig alpha, surface BCR lacking Ig alpha cytoplasmic domain were not constitutively internalized. The respective roles of Ig alpha and Ig beta subunits were then analyzed by expressing chimeric molecules containing the cytoplasmic domains of either subunits in a B cell line. Only the Ig alpha cytoplasmic domain contained an internalization signal that allowed constitutive endocytosis of Ig alpha chimeras via coated pits and accumulation in sorting-recycling endosomes. This internalization signal is contained in its immunoreceptor tyrosine-based activation motif. These results indicate that Ig alpha, through its immunoreceptor tyrosine-based activation motif, may account for the ability of IgM/IgD BCR to constitutively internalize monovalent Ags.