Inhibitory Effect of Carotenoids on the Degranulation of Mast Cells via Suppression of Antigen-induced Aggregation of High Affinity IgE Receptors

Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is critical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and β-carotene significantly inhibited the antigen-induced release of β-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-derived mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (FcϵRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited FcϵRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of FcϵRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of FcϵRI to lipid rafts, which are known as platforms of the aggregation of FcϵRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of FcϵRI to lipid rafts. This is the first report that focused on the aggregation of FcϵRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts.Mast cells play pivotal roles in inflammation and immediate-type allergic reactions by secreting biologically active substances including histamine, eicosanoids, proteolytic enzymes, cytokines, and chemokines. The antigen-induced aggregation of the high affinity IgE receptor (FcϵRI)² expressed on the cell surface triggers the degranulation of mast cells. FcϵRI has a tetrameric structure comprised of an IgE binding α-chain, a β-chain, and a disulfide-linked γ-chain dimer (1). The aggregation of FcϵRI by means of multivalent antigen-IgE complexes activates cytosolic Src protein-tyrosine kinases, such as Fyn and Lyn, which then regulate the activation of mast cells (2). Fyn kinase plays a key role in mast cell degranulation and in cytokine production by regulating Gab2 and phosphatidylinositol 3-kinase (3). Phosphorylated Lyn activates immunoreceptor tyrosine-based activation motifs of the β- and γ-chains, and the phosphorylated immunoreceptor tyrosine-based activation motifs of the γ-chain phosphorylate Syk kinase. Thereafter, a number of other signaling and adaptor molecules, such as phospholipase Cγ and protein kinase C (PKC), are phosphorylated (4). Phospholipase Cγ catalyzes the generation both of inositol 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5-trisphosphate is an inducer of intracellular Ca²⁺ mobilization, which is critical for degranulation, and diacylglycerol is an activator of PKC (5). Activated PKC is translocated from the cytosol to the plasma membrane fraction. PKC regulates many functions of mast cells, including leukotriene generation, cytokine synthesis, and degranulation (6, 7).Many studies have provided evidence that lipid rafts are involved in the activation of intracellular signaling molecules mediated by FcϵRI, the T cell receptor, the B cell receptor, and other cell surface receptors (8, 9). Lipid rafts are originally defined as microdomains in terms of their resistance to solubilization by non-ionic detergents such as Triton X-100, and are enriched in sphingolipids and cholesterol (10). Because numerous cell surface receptors and palmitoyl-anchored signaling molecules, including Src family tyrosine kinases, are associated with lipid rafts, it has been suggested that lipid rafts function as platforms regulating the induction of signaling pathways. Aggregated, but not non-aggregated, FcϵRIs are localized in lipid rafts fractionated by sucrose density gradient ultracentrifugation of detergent-treated cells (11, 12). The translocation of FcϵRI to lipid rafts is the key event that initiates the degranulation.Carotenoids are a class of widespread natural pigments that have multiple functions (13). Dietary carotenoids have been associated with a decreased risk for certain types of immune diseases, such as asthma and atopic dermatitis. Consumption of β-carotene suppresses the production of specific IgE and IgG₁ and decreases antigen-induced anaphylactic responses due to an improvement of the T_h1-T_h2 balance (14). Furthermore, β-carotene blocks nuclear translocation of the NF-κB p65 subunit, which correlates with the prevention of IκBα phosphorylation and degradation (15). It has been reported that fucoxanthin, a major carotenoid of edible brown algae, shows an anti-inflammatory effect on endotoxin-induced uveitis by decreasing the production of prostaglandin E₂ and tumor necrosis factor-α (16). Astaxanthin, found in the red pigment of crustacean shells and salmon, also has anti-inflammatory effects due to its suppression of NF-κB activation (17, 18). It has been assumed that these anti-inflammatory activities of carotenoids are due to their antioxidant activity. However, there is no information to date about the direct effect of carotenoids on the degranulation of mast cells.In the present study, we investigated the effects of carotenoids on antigen-induced degranulation of RBL-2H3 cells and mouse bone marrow-derived mast cells. In addition, to elucidate the mechanism of the modulation of degranulation by carotenoids, we focused on FcϵRI-mediated signaling in mast cells.     

Localized Diacylglycerol-dependent Stimulation of Ras and Rap1 during Phagocytosis

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcγ receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcγ receptor-mediated phagocytosis to enhance the innate immune response.Receptors that interact with the constant region of IgG (FcγR)⁴ mediate the recognition and elimination of soluble immune complexes and particles coated (opsonized) with immunoglobulins. Clustering of FcγR on the surface of leukocytes upon attachment to multivalent ligands induces their activation and subsequent internalization. Soluble immune complexes are internalized by endocytosis, a clathrin- and ubiquitylation-dependent process (1). In contrast, large, particulate complexes like IgG-coated pathogens are ingested by phagocytosis, a process that is contingent on extensive actin polymerization that drives the extension of pseudopods (2). In parallel with the internalization of the opsonized targets, cross-linking of phagocytic receptors triggers a variety of other responses that are essential components of the innate immune response. These include degranulation, activation of the respiratory burst, and the synthesis and release of multiple inflammatory agents (3, 4).Like T and B cell receptors, FcγR possesses an immunoreceptor tyrosine-based activation motif that is critical for signal transduction (3, 4). Upon receptor clustering, tyrosyl residues of the immunoreceptor tyrosine-based activation motif are phosphorylated by Src family kinases, thereby generating a docking site for Syk, a tyrosine kinase of the ZAP70 family (3, 4). The recruitment and activation of Syk in turn initiates a cascade of events that include activation of Tec family kinases, Rho- and ARF-family GTPases, phosphatidylinositol 3-kinase, phospholipase Cγ (PLCγ), and a multitude of additional effectors that together remodel the underlying cytoskeleton, culminating in internalization of the bound particle (5, 6).Phosphoinositide metabolism is thought to be critical for FcγR-induced phagocytosis (7, 8). Highly localized and very dynamic phosphoinositide changes have been observed at sites of phagocytosis: phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) undergoes a transient accumulation at the phagocytic cup, which is rapidly superseded by its complete elimination from the nascent phagosome (7). The secondary disappearance of PtdIns(4,5)P₂ is attributable in part to the localized generation of phosphatidylinositol 3,4,5-trisphosphate, which has been reported to accumulate at sites of phagocytosis (9). Activation of PLCγ is also believed to contribute to the acute disappearance of PtdIns(4,5)P₂ in nascent phagosomes. Indeed, the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate has been detected by chemical means during FcγR-evoked particle ingestion (10, 11). Moreover, imaging experiments revealed that DAG appears at the time and at the precise site where PtdIns(4,5)P₂ is consumed (7).Two lines of evidence suggest that the DAG generated upon engagement of phagocytic receptors modulates particle engulfment. First, antagonists of PLC severely impair phagocytosis by macrophages (7, 12). This inhibition is not mimicked by preventing the associated [Ca²⁺] transient, suggesting that DAG, and not inositol 1,4,5-trisphosphate, is the crucial product of the PLC (13). Second, the addition of exogenous DAG or phorbol esters, which mimic the actions of endogenous DAG, augment phagocytosis (14, 15).Selective recognition of DAG by cellular ligands is generally mediated by specific regions of its target proteins, called C1 domains (16). Proteins bearing C1 domains include, most notably, members of the classical and novel families of protein kinase C (PKC), making them suitable candidates to account for the DAG dependence of phagocytosis. Indeed, PKCα, a classical isoform, and PKCϵ and PKCδ, both novel isoforms, are recruited to phagosomes (12, 15, 17, 18). Although the role of the various PKC isoforms in particle engulfment has been equivocal over the years, Cheeseman et al. (12) convincingly demonstrated that PKCϵ contributes to particle uptake in a PLC- and DAG-dependent manner.PKCs are not the sole proteins bearing DAG-binding C1 domains. Similar domains are also found in several other proteins, including members of the RasGRP family, chimaerins, and Munc-13 (19–21). One or more of these could contribute to the complex set of responses elicited by FcγR-induced DAG production. The RasGRP proteins are a class of exchange factors for the Ras/Rap family of GTPases (22). There are four RasGRP proteins (RasGRP1 to -4), and emerging evidence has implicated RasGRP1 and RasGRP3 in T and B cell receptor signaling (23–27).The possible role of DAG-mediated signaling pathways other than PKC in phagocytosis and the subsequent inflammatory response has not been explored. Here, we provide evidence that DAG stimulates Ras and Rap1 at sites of phagocytosis, probably through RasGRPs. Last, the functional consequences of Ras and Rap1 activation were analyzed.     

Cbl Enforces an SLP76-dependent Signaling Pathway for T Cell Differentiation

A signaling pathway involving ZAP-70, LAT, and SLP76 has been regarded as essential for receptor-driven T cell development and activation. Consistent with this model, mice deficient in SLP76 have a complete block at the double negative 3 stage of T cell development. Recently, however, it has been reported that inactivation of Cbl, a ubiquitin-protein isopeptide ligase, partially rescues T cell development in SLP76-deficient mice. To probe the influence of Cbl on domain-specific SLP76 functions, we reconstituted SLP76^-/- Cbl^-/- mice with Slp76 transgenes bearing mutations in each of three functional domains of SLP76 as follows: Y3F, in which the amino-terminal tyrosine residues of SLP76 were mutated, eliminating sites of SLP76 interaction with Vav, Nck, and Itk; Δ20, in which 20 amino acids in the proline-rich region of SLP76 were deleted, removing a binding site for Gads; and RK, in which arginine 448 of SLP76 was replaced by lysine, abolishing function of the Src homology 2 domain. Although each of these transgenes has been shown to partially rescue T cell development in SLP76^-/- mice, we report here that Cbl inactivation completely reverses the severe double negative 3 developmental block that occurs in SLP76-deficient mice expressing the Y3F transgene (Y3F mice) and partially rescues the defect in positive selection in T cell receptor transgenic Y3F mice, but in contrast fails to rescue thymic development of SLP76-deficient mice expressing the Δ20 or RK transgene. Rescue in SLP76^-/-Cbl^-/-Y3F double-positive thymocytes is associated with enhanced tyrosine phosphorylation of signaling molecules, including Lck, Vav, PLC-γ1, and ERKs, but not Itk, in response to T cell receptor stimulation. Thus, our data demonstrate that Cbl suppresses activation of a bypass signaling pathway and thereby enforces SLP76 dependence of early T cell development.T cell development proceeds through multiple stages that regulate the generation and selection of T cells whose T cell receptors (TCR)² have an appropriate range of affinity for peptides presented by major histocompatibility complex (MHC) molecules (1). Precursors give rise to immature CD4^-CD8^- double negative (DN) cells that can be further divided into DN1, DN2, DN3, and DN4 stages, distinguished by cell surface phenotype as well as by critical events, including expansion of DN3 cells that have successfully rearranged TCRβ and have expressed and signaled through the pre-TCR complex (2). DN3 cells differentiate to the DN4 and then CD4⁺CD8⁺ double-positive (DP) stage following pre-TCR signaling. DP thymocytes rearrange TCRα, express a mature TCRαβ receptor, and develop into mature CD4⁺CD8^- or CD4^-CD8⁺ single-positive (SP) cells through a process of positive and negative selection that is based on signaling through this mature TCR and selection of a T cell repertoire that is tolerant to self but capable of responding to foreign-peptide-MHC (pMHC) complexes (1, 3, 4). Finally, SP cells exit from the thymus as mature T cells capable of recognizing and responding to foreign antigens.The signals from pre-TCR and TCR, which determine the fate of developing thymocytes, have been intensely studied. Ligation of the TCR by pMHC complexes results in activation of a signaling cascade initiated by phosphorylation and activation of TCR-ζ, Lck, and ZAP-70, which in turn phosphorylate downstream targets, including LAT and SLP76. ZAP-70, LAT and SLP76 proteins (3) have been shown to be essential for thymocyte development by studies, including genetic manipulation in mice (5–8). There are essentially no detectable DP or SP thymocytes or peripheral T cells in LAT^-/- or SLP76^-/- mice, in which thymocyte development is blocked at the DN3 stage (5, 7). ZAP70^-/- thymocytes are blocked at the DP stage of T cell development, and ZAP70^-/- mice have very few SP thymocytes or peripheral T cells (6). These studies suggest that signal transduction required for early T cell development proceeds through a pathway that involves critical roles of multiple molecules, including ZAP-70, LAT, and SLP76.SLP76 consists of three functional domains as follows: an amino-terminal domain containing targets for tyrosine phosphorylation, a proline-rich region, and a carboxyl-terminal SH2 domain (9). The amino-terminal tyrosine residues (Tyr-112, Tyr-128, and Tyr-145) are phosphorylated by tyrosine kinases following TCR engagement, enabling SLP76 to interact with Vav, a Rho guanine nucleotide exchange factor, Nck, an adaptor protein, and Itk, a member of Tec family PTK. The proline-rich region of SLP76 has the capacity to bind Gads, a Grb2 homolog, which results in the recruitment of SLP76 to cell surface membrane lipid rafts through binding to LAT following TCR engagement. The carboxyl-terminal SH2 domain of SLP76 interacts with ADAP (adhesion and degranulation-promoting protein) (10) an adaptor protein, and HPK-1, a serine kinase (9). Reconstitution of SLP76-deficient mice with transgenes containing mutations in each of these domains has demonstrated that each region is required for normal thymocyte development (5, 8). Two groups have reconstituted SLP76-deficient mice with T cell-specific expression of wild-type and mutant SLP76 transgenes, including a mutant in which three tyrosine residues (Tyr-112, Tyr-128, and Tyr-145) in the amino-terminal domain of SLP76 were substituted by phenylalanines (Y3F); a mutant in which 20 amino acids (amino acids 224–244) in the proline-rich region of SLP76 were deleted (Δ20); and a mutant in which arginine 448 of SLP76 was replaced by lysine (RK) (11, 12). The profound defects in T cell development and activation that are observed in SLP76 knock-out mice are completely reversed by reconstitution with a wild-type SLP76 transgene. In contrast, however, reconstitution with SLP76 that has been mutated in any of its three functional domains only partially rescues T cell development in SLP76 knock-out mice.c-Cbl (Cbl) is a ubiquitin ligase and adaptor protein (regulator) with multiple domains that associate with multiple molecules involved in signal transduction (13). Thymocytes from Cbl knock-out mice have enhanced cell surface expression of TCR and CD3 in comparison with control mice (14, 15). In addition, it has been observed that phosphorylation of ZAP-70, LAT, and SLP76 is increased in Cbl^-/- mouse thymocytes (14, 15). Recently, we reported that inactivation of Cbl partially rescues T cell development in LAT and SLP76-deficient mice (16), and Myers et al. (17) reported that inactivation of Cbl partially rescues T cell development in ZAP-70-deficient mice. These observations indicate that Cbl mediates requirements for LAT, SLP76, and ZAP-70 by preventing signaling that is capable of supporting T cell differentiation independent of LAT, SLP76, or ZAP-70. However, the rescue of T cell development in these model systems is strikingly incomplete, failing to substantially reconstitute development through the pre-TCR-dependent DN3-DN4 transition and thus failing to generate normal numbers of DP or functionally mature SP thymocytes. These findings suggest that Cbl inactivation functions to enable pathways that are capable of bypassing some but not all of the requirements for ZAP-70, LAT, and SLP76 during T cell development. To define these signaling pathways, normally suppressed by Cbl, that can support T cell development, we assessed the ability of Cbl inactivation to rescue T cell development in the presence of Y3F, Δ20, or RK SLP76 mutant transgenes. In this study, we report that Cbl inactivation completely reverses the DN3-DN4 developmental defect and partially reverses alterations in positive selection in thymocytes of SLP76 knock-out mice reconstituted with the SLP76 mutant Y3F, which lacks amino-terminal phosphotyrosine residues. In contrast, Cbl inactivation has no effect on the thymic developmental defects observed in SLP76 knock-out mice reconstituted with Slp76 transgenes mutated in the proline-rich Gads-binding region (Δ20) or the carboxyl-terminal SH2 domain (RK). Biochemical studies revealed that rescue of development in SLP76^-/-Y3F thymocytes by inactivation of Cbl was marked by reversal of defects in tyrosine phosphorylation of multiple molecules, including Lck, Vav, PLC-γ1, and ERKs in response to TCR stimulation of DP thymocytes. Thus, Cbl normally enforces SLP76 dependence of T cell development by inhibiting an alternative pathway that may be independent of SLP76 association with Vav, Nck, and Itk (18).     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Independent and Cooperative Roles of Adaptor Molecules in Proximal Signaling during Fc?RI-Mediated Mast Cell Activation

Activation through FcɛRI, a high-affinity IgE-binding receptor, is critical for mast cell function during allergy. The formation of a multimolecular proximal signaling complex nucleated by the adaptor molecules SLP-76 and LAT1 is required for activation through this receptor. Based on previous T-cell studies, current dogma dictates that LAT1 is required for plasma membrane recruitment and function of SLP-76. Unexpectedly, we found that the recruitment and phosphorylation of SLP-76 were preserved in LAT1^−/− mast cells and that SLP-76^−/− and LAT1^−/− mast cells harbored distinct functional and biochemical defects. The LAT1-like molecule LAT2 was responsible for the preserved membrane localization and phosphorylation of SLP-76 in LAT1^−/− mast cells. Although LAT2 supported SLP-76 phosphorylation and recruitment to the plasma membrane, LAT2 only partially compensated for LAT1-mediated cell signaling due to its decreased ability to stabilize interactions with phospholipase Cγ (PLCγ). Comparison of SLP-76^−/− LAT1^−/− and SLP-76^−/− mast cells revealed that some functions of LAT1 could occur independently of SLP-76. We propose that while SLP-76 and LAT1 depend on each other for many of their functions, LAT2/SLP-76 interactions and SLP-76-independent LAT1 functions also mediate a positive signaling pathway downstream of FcɛRI in mast cells.Mast cell activation during allergic inflammation is mediated by the high-affinity immunoglobulin E (IgE)-binding receptor FcɛRI. Cross-linking of FcɛRI on mast cells by IgE/cognate antigen complexes results in the rapid release of a wide array of inflammatory mediators, including vasoactive amines and cytokines/chemokines that give rise to allergic symptoms, ranging in severity from simple urticaria to anaphylactic shock and death (14). As allergy affects ∼30% of the population in developed countries (13), much attention has been placed on studying the signal transduction mechanisms involved in mast cell activation downstream of FcɛRI in hopes of finding novel targets for therapeutic intervention.Signal transduction downstream of FcɛRI is initiated by the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) contained in the signaling components (β and γ chains) of the FcɛRI complex (30, 37). Once phosphorylated, these chains serve as docking sites for several protein tyrosine kinases (PTKs), including Lyn and spleen tyrosine kinase (Syk) (9, 19, 34). Recruitment of Syk to the membrane by FcɛRI results in the phosphorylation of scaffold proteins known as adaptor molecules. Adaptor proteins lack enzymatic activity but instead contain protein-binding domains that are critical for the formation of a multimolecular complex, which orchestrates downstream signaling in a temporal and spatial manner. The adaptor molecules Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) and linker of activated T cells 1 (LAT1) organize the assembly of a proximal signaling complex downstream of FcɛRI. Failure to form this complex is detrimental to FcɛRI-mediated mast cell function, as demonstrated by the finding that both SLP-76-deficient (22, 29, 41) and LAT1-deficient (25, 31, 32) mast cells display severely diminished degranulation and cytokine/chemokine production following FcɛRI ligation.Similar proximal signaling complexes are formed downstream of several different ITAM-containing receptors. Much of our understanding of the role of adaptor molecules in signal transduction has come from identification of phosphoproteins during T-cell receptor (TCR)-mediated activation of the human Jurkat T-cell line (1, 33). These studies eventually led to a paradigm describing the sequence of events in the formation of the SLP-76/LAT1 signaling complex. According to this model, SLP-76 is found constitutively bound to Grb2-related adaptor downstream of Shc (GADS) (24) and resides in the cytosol. Upon TCR activation, the tyrosines of membrane-resident LAT1 are phosphorylated and become attachment sites for proteins such as phospholipase Cγ (PLCγ) and GADS (43, 45). SLP-76 is drawn to the membrane through a GADS/LAT1 interaction, which then permits Syk family PTKs to maximally phosphorylate the N-terminal tyrosines of SLP-76 (5, 10). Several lines of evidence support this model whereby a LAT1/SLP-76 module organizes TCR signaling. First, both SLP-76- and LAT1-deficient Jurkat T cells display similar biochemical defects, such as diminished PLCγ and extracellular signal-regulated kinase (ERK) activation (10, 42). Second, T cells in SLP-76^−/− and LAT1^−/− mice are blocked at the same stage of development (7, 44). Third, SLP-76 can be coimmunoprecipitated with LAT1 but not with LAT1 harboring tyrosine-to-phenylalanine mutations (45). Finally, expression of a fusion protein comprised of the membrane-localizing domain of LAT1 and SLP-76 that forces localization of SLP-76 to the plasma membrane rescues the TCR-induced functional defects of both SLP-76- and LAT1-deficient Jurkat T cells (3). This model implies a mutually dependent relationship between SLP-76 and LAT1, where SLP-76 and LAT1 rely on each other to carry out their roles.One might suspect that this model for LAT1/SLP-76 function would operate in all other cells that utilize these adaptor molecules for ITAM-containing receptor-mediated signaling. However, the published defects of LAT1-deficient mast cells in FcɛRI-mediated signaling appeared milder than those of SLP-76-deficient mast cells, although a direct comparison has never been reported. In the present study, we show that LAT1-deficient mast cells display distinct functional and biochemical defects compared to SLP-76-deficient mast cells, implying that unlike in T cells, SLP-76 may not depend entirely on LAT1 for its function in mast cells. Surprisingly, the membrane recruitment and phosphorylation of SLP-76 were also preserved in LAT1^−/− mast cells. We show that LAT2 (also known as non-T-cell activation linker [NTAL] or linker for activation of B cells [LAB]), which is not expressed in naïve T cells but is expressed in mast cells (15), is responsible for phosphorylation and plasma membrane recruitment of SLP-76 in the absence of LAT1. However, LAT2 cannot support all LAT1/SLP-76-associated functions, such as sustained Ca²⁺ flux, likely due to decreased stability of the LAT2/SLP-76/PLCγ complex. Comparison of SLP-76^−/− LAT1^−/− and SLP-76^−/− mast cells also revealed that some functions of LAT1 could occur independently of SLP-76. We propose that although SLP-76 and LAT1 are interdependent for many of their functions, LAT2/SLP-76 interactions and SLP-76-independent LAT1 functions mediate positive signaling downstream of FcɛRI in mast cells.     

Infection of Glial Cells by the Human Polyomavirus JC Is Mediated by an N-Linked Glycoprotein Containing Terminal α(2-6)-Linked Sialic Acids

The human JC polyomavirus (JCV) is the etiologic agent of the fatal central nervous system (CNS) demyelinating disease progressive multifocal leukoencephalopathy (PML). PML typically occurs in immunosuppressed patients and is the direct result of JCV infection of oligodendrocytes. The initial event in infection of cells by JCV is attachment of the virus to receptors present on the surface of a susceptible cell. Our laboratory has been studying this critical event in the life cycle of JCV, and we have found that JCV binds to a limited number of cell surface receptors on human glial cells that are not shared by the related polyomavirus simian virus 40 (C. K. Liu, A. P. Hope, and W. J. Atwood, J. Neurovirol. 4:49–58, 1998). To further characterize specific JCV receptors on human glial cells, we tested specific neuraminidases, proteases, and phospholipases for the ability to inhibit JCV binding to and infection of glial cells. Several of the enzymes tested were capable of inhibiting virus binding to cells, but only neuraminidase was capable of inhibiting infection. The ability of neuraminidase to inhibit infection correlated with its ability to remove both α(2-3)- and α(2-6)-linked sialic acids from glial cells. A recombinant neuraminidase that specifically removes the α(2-3) linkage of sialic acid had no effect on virus binding or infection. A competition assay between virus and sialic acid-specific lectins that recognize either the α(2-3) or the α(2-6) linkage revealed that JCV preferentially interacts with α(2-6)-linked sialic acids on glial cells. Treatment of glial cells with tunicamycin, but not with benzyl N-acetyl-α-d-galactosaminide, inhibited infection by JCV, indicating that the sialylated JCV receptor is an N-linked glycoprotein. As sialic acid containing glycoproteins play a fundamental role in mediating many virus-cell and cell-cell recognition processes, it will be of interest to determine what role these receptors play in the pathogenesis of PML.Approximately 70% of the human population worldwide is seropositive for JC virus (JCV). Like other polyomaviruses, JCV establishes a lifelong latent or persistent infection in its natural host (40, 49, 50, 68, 72). Reactivation of JCV in the setting of an underlying immunosuppressive illness, such as AIDS, is thought to lead to virus dissemination to the central nervous system (CNS) and subsequent infection of oligodendrocytes (37, 40, 66, 68). Reactivation of latent JCV genomes already present in the CNS has also been postulated to contribute to the development of progressive multifocal leukoencephalopathy (PML) following immunosuppression (19, 48, 55, 70, 75). Approximately 4 to 6% of AIDS patients will develop PML during the course of their illness (10). In the CNS, JCV specifically infects oligodendrocytes and astrocytes. Outside the CNS, JCV genomes have been identified in the urogenital system, in the lymphoid system, and in B lymphocytes (2, 17, 18, 30, 47, 59). In vitro, JCV infects human glial cells and, to a limited extent, human B lymphocytes (3, 4, 39, 41, 42). Recently, JCV infection of tonsillar stromal cells and CD34⁺ B-cell precursors has been described (47). These observations have led to the suggestion that JCV may persist in a lymphoid compartment and that B cells may play a role in trafficking of JCV to the CNS (4, 30, 47).Virus-receptor interactions play a major role in determining virus tropism and tissue-specific pathology associated with virus infection. Viruses that have a very narrow host range and tissue tropism, such as JCV, are often shown to interact with high affinity to a limited number of specific receptors present on susceptible cells (26, 44). In some instances, virus tropism is strictly determined by the presence of specific receptors that mediate binding and entry (7, 16, 27, 35, 46, 53, 56, 67, 73, 74, 76). In other instances, however, successful entry into a cell is necessary but not sufficient for virus growth (5, 8, 45, 57). In these cases, additional permissive factors that interact with viral regulatory elements are required.The receptor binding characteristics of several polyomaviruses have been described. The mouse polyomavirus (PyV) receptor is an N-linked glycoprotein containing terminal α(2-3)-linked sialic acid (12–14, 22, 28). Both the large and small plaque strains of PyV recognize α(2-3)-linked sialic acid. The small-plaque strain also recognizes a branched disialyl structure containing α(2-3)- and α(2-6)-linked sialic acids. Neither strain recognizes straight-chain α(2-6)-linked sialic acid. The ability of the large- and small-plaque strains of PyV to differentially recognize these sialic acid structures has been precisely mapped to a single amino acid in the major virus capsid protein VP1 (21). The large-plaque strains all contain a glycine at amino acid position 92 in VP1, and the small-plaque strains all contain a negatively charged glutamic acid at this position (21). In addition to forming small or large plaques, these strains also differ in the ability to induce tumors in mice (20). This finding suggests that receptor recognition plays an important role in the pathogenesis of PyV.The cell surface receptor for lymphotropic papovavirus (LPV) is an O-linked glycoprotein containing terminal α(2-6)-linked sialic acid (26, 33, 34). Infection with LPV is restricted to a subset of human B-cell lines, and recognition of specific receptors is a major determinant of the tropism of LPV for these cells (26).Unlike the other members of the polyomavirus family, infection of cells by simian virus 40 (SV40) is independent of cell surface sialic acids. Instead, SV40 infection is mediated by major histocompatibility complex (MHC)-encoded class I proteins (5, 11). MHC class I proteins also play a role in mediating the association of SV40 with caveolae, a prerequisite for successful targeting of the SV40 genome to the nucleus of a cell (1, 63). Not surprisingly, SV40 has been shown not to compete with the sialic acid-dependent polyomaviruses for binding to host cells (15, 26, 38, 58).Very little is known about the early steps of JCV binding to and infection of glial cells. Like other members of the polyomavirus family, JCV is known to interact with cell surface sialic acids (51, 52). A role for sialic acids in mediating infection of glial cells has not been described. It is also not known whether the sialic acid is linked to a glycoprotein or a glycolipid. In a previous report, we demonstrated that JCV bound to a limited number of cell surface receptors on SVG cells that were not shared by the related polyomavirus SV40 (38). In this report, we demonstrate that virus binding to and infection of SVG cells is dependent on an N-linked glycoprotein containing terminal α(2-3)- and α(2-6)-linked sialic acids. Competitive binding assays with sialic acid-specific lectins suggest that the virus preferentially interacts with α(2-6)-linked sialic acids. We are currently evaluating the role of this receptor in determining the tropism of JCV for glial cells and B cells.     

Syk Tyrosine 317 Negatively Regulates Osteoclast Function via the Ubiquitin-Protein Isopeptide Ligase Activity of Cbl

Cytoskeletal organization of the osteoclast (OC), which is central to the capacity of the cell to resorb bone, is induced by occupancy of the αvβ3 integrin or the macrophage colony-stimulating factor (M-CSF) receptor c-Fms. In both circumstances, the tyrosine kinase Syk is an essential signaling intermediary. We demonstrate that Cbl negatively regulates OC function by interacting with Syk^Y317. Expression of nonphosphorylatable Syk^Y317F in primary Syk^−/− OCs enhances M-CSF- and αvβ3-induced phosphorylation of the cytoskeleton-organizing molecules, SLP76, Vav3, and PLCγ2, to levels greater than wild type, thereby accelerating the resorptive capacity of the cell. Syk^Y317 suppresses cytoskeletal organization and function while binding the ubiquitin-protein isopeptide ligase Cbl. Consequently, Syk^Y317F abolishes M-CSF- and integrin-stimulated Syk ubiquitination. Thus, Cbl/Syk^Y317 association negatively regulates OC function and therefore is essential for maintenance of skeletal homeostasis.OCs² are multinucleated cells generated by fusion of mononuclear progenitors of the monocyte/macrophage family under the aegis of M-CSF and receptor activator of nuclear factor κB ligand (RANKL) (1). Upon mineralized matrix recognition, the OC polarizes its fibrillar actin, eventuating in the formation of an acidified extracellular microenvironment that degrades bone. Failure to undergo this polarization event results in OC hypo-function and consequently in varying degrees of osteopetrosis (2).Integrins are transmembrane α/β heterodimers that mediate cell-cell and cell-matrix interactions and generate intracellular signals when occupied by ligands (3). The integrin, αvβ3, is expressed by OCs, and binding of this complex to bone is pivotal to the resorptive process (4).M-CSF recognizes its transmembrane receptor tyrosine kinase, c-Fms, and induces receptor autophosphorylation at seven tyrosine residues within the cytoplasmic domain (5). Several Src homology-2 domain-containing molecules are recruited to the phosphotyrosine residues upon M-CSF binding and initiate signaling cascades that lead to cytoskeletal organization, survival, and proliferation of OC lineage cells (5–7). Both the αvβ3 integrin and M-CSF are important regulators of OC actin remodeling (4, 6, 8).Syk is a 72-kDa nonreceptor tyrosine kinase, which mediates αvβ3- and c-Fms-induced OC cytoskeletal organization and function in a phosphorylation-dependent manner via a process involving activation of associated adaptor proteins, such as SLP-76 and Vav3 (9, 10). A number of Syk tyrosine residues undergo phosphorylation following engagement of the integrin and Fcγ receptor in immune (11) and mast cells (12). Three conserved tyrosine residues in the Syk linker region, namely Tyr³¹⁷, Tyr³⁴², and Tyr³⁴⁶, lie within consensus sequences for recognition by Src homology 2 domains, suggesting they transduce signals. Although phospho-Syk^Y342 and phospho-Syk^Y346 may serve as positive signaling regulators (12–14), phosphorylation of Syk^Y317 creates a binding site for c-Cbl, an E3 ubiquitin ligase proposed to prompt ubiquitination and subsequent degradation of Syk (15, 16). Hence, Syk^Y317 is a candidate negative regulatory site, but its role in OC function and/or differentiation is unknown.Cbl is a 120-kDa protein that is tyrosine-phosphorylated following activation by growth factors, cytokines, and integrins. It has two distinct but related activities, serving both as an adaptor protein (17, 18) and E3 ubiquitin ligase (19, 20). Cbl functions principally as an adaptor in OCs by participating in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton (18, 21). In other cell types, Cbl is also a negative regulator of receptor and nonreceptor tyrosine kinases, as it promotes their degradation (22). OCs and their precursors express c-Cbl and another family member Cbl-b that compensates for the absence of c-Cbl (23, 24). As combined deletion of both isoforms eventuates in early embryonic lethality (24), it is not clear if c-Cbl functions as an E3 ubiquitin ligase in OCs. We establish that c-Cbl, recognizing Syk^Y317, prompts the ubiquitination of the kinases thereby arresting activation of cytoskeleton-organizing molecules and thus OC function. The Cbl-Syk^Y317 complex is therefore important in maintenance of normal skeletal mass.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Neutralization Sensitivity of Human Immunodeficiency Virus Type 1 Primary Isolates to Antibodies and CD4-Based Reagents Is Independent of Coreceptor Usage

We have investigated whether the identity of the coreceptor (CCR5, CXCR4, or both) used by primary human immunodeficiency virus type 1 (HIV-1) isolates to enter CD4⁺ cells influences the sensitivity of these isolates to neutralization by monoclonal antibodies and CD4-based agents. Coreceptor usage was not an important determinant of neutralization titer for primary isolates in peripheral blood mononuclear cells. We also studied whether dualtropic primary isolates (able to use both CCR5 and CXCR4) were differentially sensitive to neutralization by the same antibodies when entering U87MG-CD4 cells stably expressing either CCR5 or CXCR4. Again, we found that the coreceptor used by a virus did not greatly affect its neutralization sensitivity. Similar results were obtained for CCR5- or CXCR4-expressing HOS cell lines engineered to express green fluorescent protein as a reporter of HIV-1 entry. Neutralizing antibodies are therefore unlikely to be the major selection pressure which drives the phenotypic evolution (change in coreceptor usage) of HIV-1 that can occur in vivo. In addition, the increase in neutralization sensitivity found when primary isolates adapt to growth in transformed cell lines in vitro has little to do with alterations in coreceptor usage.Human immunodeficiency virus type 1 (HIV-1) enters CD4⁺ T cells via an interaction with CD4 and coreceptor molecules, the most important of which yet identified are the chemokine receptors CXCR4 and CCR5 (4, 12, 23, 26, 28, 32). CXCR4 is used by T-cell line-tropic (T-tropic) primary isolates or T-cell line-adapted (TCLA) lab strains, whereas CCR5 is used by primary isolates of the macrophage-tropic (M-tropic) phenotype (4, 12, 23, 26, 28, 32). Most T-tropic isolates and some TCLA strains are actually dualtropic in that they can use both CXCR4 and CCR5 (and often other coreceptors such as CCR3, Bonzo/STRL33, and BOB/gpr15), at least in coreceptor-transfected cells (18, 24, 30, 54, 89). The M-tropic and T-tropic/dualtropic nomenclature has often been used interchangeably with the terms “non-syncytium-inducing” (NSI) and “syncytium-inducing” (SI), although it is semantically imprecise to do so.M-tropic viruses are those most commonly transmitted sexually (3, 33, 87, 106) and from mother to infant (2, 72, 81). If T-tropic strains are transmitted, or when they emerge, this is associated with a more rapid course of disease in both adults (17, 37, 46, 51, 52, 76, 78, 82, 92, 101) and children (6, 45, 84, 90). However, T-tropic viruses emerge in only about 40% of infected people, usually only several years after infection (76, 78). A well-documented, albeit anecdotal, study found that when a T-tropic strain was transmitted by direct transfer of blood, its replication was rapidly suppressed: the T-tropic virus was eliminated from the body, and M-tropic strains predominated (20). These results suggest that there is a counterselection pressure against the emergence of T-tropic strains during the early stages of HIV-1 infection in most people. But what is this pressure?Since the M-tropic and T-tropic phenotypes are properties mediated by the envelope glycoproteins whose function is to associate with CD4 and the coreceptors, a selection pressure differentially exerted on M- and T-tropic viruses could, in principle, act at the level of virus entry. In other words, neutralizing antibodies to the envelope glycoproteins, or the chemokine ligands of the coreceptors, could theoretically interfere more potently with the interactions of T-tropic strains with CXCR4 than with M-tropic viruses and CCR5. A differential effect of this nature could suppress the emergence of T-tropic viruses. Consistent with this possibility, neutralizing antibodies are capable of preventing the CD4-dependent association of gp120 with CCR5 (42, 94, 103), and chemokines can also prevent the coreceptor interactions of HIV-1 (8, 13, 23, 28, 70).Here, we explore whether the efficiency of HIV-1 neutralization is affected by coreceptor usage. Although earlier studies have not found T-tropic strains to be inherently more neutralization sensitive than M-tropic ones (20, 40, 44), previously available reagents and techniques may not have been adequate to fully address this question. One major problem is that even single residue changes can drastically affect both antibody binding to neutralization epitopes and the HIV-1 phenotype (25, 55, 62, 67, 83, 91), and so studies using relatively unrelated viruses and a fixed antibody (polyclonal or monoclonal) preparation have two variables to contend with: the viral phenotype (coreceptor use) and the antigenic structure of the virus and hence the efficiency of the antibody-virion interaction.We have used a new experimental strategy to explore whether coreceptor usage affects neutralization sensitivity in the absence of other confounding variables: the use of dualtropic viruses able to enter CD4⁺ cells via either CCR5 or CXCR4. By using a constant HIV-1 isolate or clone and the same monoclonal antibodies (MAbs) or CD4-based reagents as neutralizing agents, we can ensure that the only variable under study in the neutralization reaction is the nature of the coreceptor used for entry. Our major conclusion is that there is no strong association between coreceptor usage and neutralization sensitivity for primary HIV-1 isolates. Independent studies have reached the same conclusion (53a, 59). The emergence of T-tropic (SI) viruses in vivo may be unlikely to be due to escape from antibody-mediated selection pressure.