Distinct structural compartmentalization of the signal transducing functions of major histocompatibility complex class II (Ia) molecules.

Class II major histocompatibility complex encoded proteins (MHC class II or Ia molecules) are principal plasma membrane proteins involved in activation of both B and T cells during antigen-driven immune responses. Recent data indicate that class II molecules are more than simply recognition elements that provide a ligand for the T cell antigen receptor. Changes in B cell physiology that follow class II binding are now recognized as being required not only for the induction of T cell activation, but also for B cell activation and proliferation. It is interesting to note that class II molecules appear to transduce signals via two distinct mechanisms depending upon the differentiative state of the B cell on which they are expressed. While one of these pathways, involving cAMP generation and protein kinase C localization in the cytoskeletal/nuclear compartment, is seen in resting B cells, the second is seen in primed B cells and involves tyrosine kinase activation, inositol lipid hydrolysis, and Ca2+ mobilization. Use of this pathway is correlated with ability of class II to transduce signals leading to B cell proliferation. To begin to address the molecular basis of this unique, activation-dependent, differential coupling of class II to signaling pathways, we conducted mutational analysis of class II structural requirements for signal transduction. Here we report that the cytoplasmic (Cy) domains of I-Ak class II molecules are not required for either receptor-mediated activation of protein tyrosine phosphorylation or Ca2+ mobilization. This is in contrast to the requirement of the Cy domain of beta chain of class II for the alternate signaling pathway and efficient antigen presentation to autoreactive T cell lines. Disparate distribution of functional motifs within the MHC class II molecules may reflect use of distinct receptor associated effector molecules to sustain different modes of signal transduction in various class II-expressing cells.

Role of 4-1BB ligand in costimulation of T lymphocyte growth and its upregulation on M12 B lymphomas by cAMP.

K46J B lymphomas express a T cell costimulatory activity that is not inhibited by CTLA-4Ig, anti-B7-1, anti-B7-2, anti-intercellular adhesion molecule 1 or antibodies to heat stable antigen. In this paper we report that this costimulatory activity is mediated at least in part by 4-1BB ligand, a member of the tumor necrosis factor (TNF) gene family that binds to 4-1BB, a T cell activation antigen with homology to the TNF/nerve growth factor receptor family. A fusion protein between 4-1BB and alkaline phosphatase (4-1BB-AP) blocks T cell activation by K46J lymphomas in both an antigen-specific system and with polyclonally (anti-CD3) activated T cells. 4-1BB-AP also blocks antigen presentation by normal spleen cells. When the antigen-presenting cells express B7 molecules as well as 4-1BB ligand, we find that B7 molecules and 4-1BB-AP both contribute to T cell activation. These data suggest that 4-1BB ligand plays an important role in costimulation of IL-2 production and proliferation by T cells. The B lymphoma M12 expresses low levels of 4-1BB-L but can be induced to express higher levels by treatment of the B cells with cAMP, which also induces B7-1 and B7-2 in these cells. Thus cAMP appears to coordinately induce several costimulatory molecules on B cells.

Translational diffusion of class II major histocompatibility complex molecules is constrained by their cytoplasmic domains.

Site-directed mutagenesis in vitro was used to introduce stop codons in the genomic DNA of the alpha and beta chains of the murine class II major histocompatibility complex antigen, I-Ak. Mutated DNA was transfected into B lymphoma cells that were then selected by neomycin resistance and for their ability to express I-Ak molecules on their plasma membrane. The translational diffusion coefficient (Dlat) of I-Ak molecules composed of a wild-type beta chain paired with an alpha chain missing either 6 or 12 amino acids from the cytoplasmic domain is on the average threefold higher than the Dlat of wild-type I-Ak molecules as measured by fluorescence photobleaching and recovery. The removal of 12 amino acids from the cytoplasmic domain of the beta chain did not change the Dlat value from that of wild-type I-Ak if the truncated beta chain was paired with a wild-type alpha chain. Removing all amino acids of the cytoplasmic domains of both the alpha and beta chains resulted in a 10-fold increase in the Dlat, the highest value for any of the truncated I-Ak molecules tested. These data indicate that the carboxy-terminal six amino acids of the cytoplasmic domain of the alpha chain and the six plasma membrane-proximal amino acids of the beta chain are important in constraining the translational diffusion of I-Ak molecules in the plasma membrane.

Effect of bacterial flora and mouse genotype (euthymic or athymic) on scrapie pathogenesis.

Euthymic and athymic female BALB/c mice, reared under either germfree or defined flora conditions, were used to investigate the pathogenesis of scrapie after intracerebral or intraperitoneal inoculation. Time in days to onset of clinical signs (Stage I), to endstage (Stage II), and the time interval between Stage I and Stage II were compared among groups. In addition, scrapie agent titers in spleen were determined at 28 and 90 days after infection, as were agent titers in spleen and brain at Stage II. Three-way analysis of variance indicated that the bacterial flora, the presence or absence of a thymus, and the route of agent inoculation interact to produce significant differences in the pathogenesis of disease. The three factors in the experimental design also influenced the spleen titers of scrapie infectivity. The variation in scrapie pathogenesis among the groups of mice is likely to be mediated by differences in their reticuloendothelial systems. These differences may alter the agent's adsorption in spleen and/or route of transport from spleen to brain.

Truncated MHC class II cytoplasmic and transmembrane domains: effect on plasma membrane expression.

Plasma membrane (PM) expression of major histocompatibility complex (MHC) class II molecule is required for the interaction of antigen (Ag) presenting cells and T lymphocytes. Class II molecules composed of an alpha and a beta chain are highly polymorphic which facilitates their interaction with Ag and Ag-specific T cells. Recently, we have focused on the less polymorphic sequences of class II molecules, the transmembrane (TM) and cytoplasmic (Cy) domains, in an attempt to understand what their function might be. Using site-directed mutagenesis to create truncations in the TM and Cy domains of IAk's alpha or beta chain, or both, we have identified some of the sequence requirements for efficient surface expression of I-Ak molecules. Ak beta TM mutants that are not expressed at the PM are not transported past the medial-Golgi as indicated by in situ staining and Western blot analysis of endoglycosidase-H-treated immunoprecipitates. The lack of transport of TM class II mutants is not due to lack of association with the invariant chain (Ii). Class II molecules with Cy domain truncations in both chains are not efficiently transported to the PM and also have a percentage of molecules that are endoglycosidase-H sensitive. In situ staining of class II in cells expressing Cy domain truncated class II molecules revealed a discrete vesicular pattern compared to the staining of transfectants that expressed wildtype class II molecules. The immunofluorescence data along with the endoglycosidase-H data indicate the Cy domains are required for efficient transport. Immunoprecipitation studies using a panel of I-Ak conformation-specific antibodies revealed that the truncation of the Cy domains of both chains did not effect the conformation of class II. However, further truncation of the Ak beta chain into the TM domain resulted in lack of transport past the ER/medial-Golgi and diminished expression (stability) of mutant class II proteins within the cells. The alpha/beta chains of the TM mutants that did associate bound a panel of conformation sensitive antibodies except for one, 3F12. We conclude that the Cy domain of the alpha and beta chains of MHC class II, as well as sequences in the TM domains of the Ak beta chain are required for efficient class II PM expression. The reason for the lack of PM expression of TM mutants may be the inability to assess a transport competent conformation as defined by the 3F12-specific epitope, while truncation of the Ak alpha Cy domains is proposed to prevent complete masking of the ER retention sequence of the Ii chain.

Dynamics of molecules involved in antigen presentation: effects of fixation.

Antigen presentation by MHC class II molecules can be enhanced by paraformaldehyde fixation of antigen-presenting cells prior to assay. This treatment might be expected to aggregate membrane proteins and thus stabilize and strengthen transient protein-protein interactions involved in intercellular cooperation. Lateral and rotational dynamics of the MHC class II antigen I-Ad on A20 cells fixed with various concentrations of paraformaldehyde were examined by fluorescence photobleaching recovery and time-resolved phosphorescence anisotropy, respectively. Probes were tetramethylrhodamine and erythrosin conjugates of MKD6 Fab fragments. Increasing concentrations of paraformaldehyde led to a progressive increase in the limiting anisotropy of I-Ad at 4 degrees C from the value of 0.042 for untreated cells, indicative of large aggregate formation, while leaving the rotational correlation time of 29 micros unchanged, a measure of the unperturbed molecule. On the other hand, the translational diffusion constants decreased from approximately 2x10(-10) cm2 s(-1), while the fractional recovery remained unchanged at about 40-50%. Taken together, these results suggest that fixation crosslinks class II molecules to each other or to other membrane proteins into structures large enough (>500,000 kDa) to diffuse translationally with perceptibly size-dependent rates. The fixation effects on both class II rotation and lateral diffusion were half-maximal at paraformaldehyde concentrations of approximately 0.2%. Possible relations between the biological effector functions of class II and the physical sizes of fixation-induced aggregates are discussed.

Lateral dynamics of major histocompatibility complex class II molecules bound with agonist peptide or altered peptide ligands.

We examined the lateral diffusion of I-Ad on A20 cells following the binding of ovalbumin-derived peptides. The peptides were OVA323-339 and OVA325-335 and a related peptide OVA325-335s substituted H331Q. Only OVA323-339 and OVA325-335 were effectively presented by A20 cells to DO-11.10/S4.4 T cells as assessed by IL-2 production. Fluorescence photobleaching recovery (FPR) measurements showed anti-I-Ad to have a lateral diffusion coefficient on untreated A20 cells of 1.8 +/- 1.0 x 10(-10) cm2 s-1 at 25 degrees C with fluorescence recovery after photobleaching greater than 50%. After 24 h incubation of A20 cells with OVA323-339 or OVA325-335, a subpopulation of A20 cells appeared that were approximately half the size of untreated A20 cells. Culture of A20 with OVA325-355s did not stimulate DO-11.10 cells or induce a size change in A20 cells. Class II molecules were laterally immobile on these small cells with fluorescence recoveries after photobleaching of less than 20%. The relative number of small cells in the A20 cell population was correlated with the immunogenicity of the peptides. These results suggest that immobilization of surface I-Ad may be an important event in antigen presentation.

Class II cytoplasmic and transmembrane domains are not required for class II-mediated B cell spreading.

B cells cultured on immobilized anti-class II monoclonal antibody (mAb) change from round to flattened cells, with lamellipodia and filopodia. This change in cell morphology, termed 'spiders', occurs within 30 min upon culture and is mediated through either I-A or I-E molecules. Class II molecules that are defective in mediating protein kinase C (PKC) due to the deletions of both alpha and beta chain's cytoplasmic (Cy) domain sequences can induce spider formation. B-cell transfectants that express chimeric MHC class II/class I molecules, where the ectodomains are class II sequences and the transmembrane and Cy domains are class I sequences also form spiders when cultured on anti-class II mAb. The spider morphology is not induced by either anti-immunoglobulin (Ig) or anti-MHC class I mAb. Treatment of B cells to increase intracellular cAMP, a component of the class II signaling pathway also results in spider formation with the same kinetics and percent change in the responding population as that induced by anti-class II mAb. Cytochalasin A treatment which disrupts cytoskeletal actin filaments and the tyrosine kinase inhibitor, genistein, both inhibit spider formation. Actin redistributes from a concentric ring in round cells to the ends of the filopodia in the spiders. The mechanism of spider induction whether resultant from second messengers following class II signaling or from non-signaling-induced physical interactions of class II with intracellular cytoskeletal components only requires the extracellular domains of class II. The biologic relevance of B-cell spiders is currently not known but has been reported to be associated with class II signal transduction and efficient Ag presentation.

Molecular dynamics of point mutated I-A(k) molecules expressed on lymphocytes.

We have recently reported the lateral and rotational diffusion parameters for I-A(k) molecules expressing various cytoplasmic truncations (Int. Immunol. 12 (2000) 1319). We now describe the membrane dynamics of I-A(k) with various mutations in the presumed contact region between alphabeta-heterodimers in an (alphabeta)2 dimer of dimers structure. Such mutations are known to strongly affect the antigen presentation ability of these molecules (Int. Immunol. 10 (1998) 1237-1249) but cause relatively small changes in the molecular dynamics of I-A(k). Lateral diffusion coefficients of I-A(k) wild-type molecules and mutants obtained via fringe fluorescence photobleaching recovery (FPR) ranged from 1.1 to 2.3x10(-10)cm2/s at room temperature while fractional mobilities averaged 75+/-6%. For all cell types examined, treatment with either hen egg lysozyme 46-61 peptide or db-cAMP reduced the I-A(k) mobile fraction by about 10% relative to untreated cells, suggesting that these treatments may increase lateral confinement of class II in lipid rafts or cytoskeletal interactions of the molecules. Wild-type I-A(k) and mutants capable of normal or partial antigen presentation exhibited, as a group, slightly longer rotational correlation times (RCT) at 4 degrees C than did mutants inactive in antigen presentation, 14+/-4 versus 10+/-1 micros, respectively. Moreover, peptide, cAMP and anti-CD40 mAb treatment all increased rotational correlation times for fully- and partially-functional I-A(k) but not for non-functional molecules. For example, 16 h peptide treatment yielded average RCTs of 28+/-12 and 10+/-1 micros for the groups of functional and non-functional molecules, respectively. Such modulation of the dynamics of functional class II molecules is consistent with these treatments' stabilization of class II or induction of new gene expression. Measurements of fluorescence resonant energy transfer between I-A(k), though complicated by cellular autofluorescence, averaged 6+/-7% over 15 cells or treatments, a result consistent with the presence of a small fraction of I-A(k) as a dimer of dimers species. In summary, our results suggest subtle changes in the molecular motions of class II molecules correlate with a significant impact on class II function. Molecules active in antigen presentation exhibit more restricted motion in the membrane, and thus presumably more extensive intermolecular interactions, than non-functional molecules. Further, treatments, such as db-cAMP and anti-CD40, which rescue antigen presentation by partially defective mutants, appear to increase such interactions, several types of which have already been reported for class II. A more detailed understanding of these phenomena will require both more sensitive biophysical tools and a more refined model of the role of class II intermolecular interactions in antigen presentation.

Salt bridge residues between I-Ak dimer of dimers alpha-chains modulate antigen presentation.

Class II dimers of dimers are predicted to have functional significance in antigen presentation. The putative contact amino acids of the I-Ak class II dimer of dimers have been identified by molecular modeling based on the DR1 crystal structure (Nydam et al., Int. Immunol. 10, 1237,1998). We have previously reported the role in antigen presentation of dimer of dimers contact amino acids located in the C-terminal domains of the alpha- and beta-chains of class II. Our calculations show that residues Ealpha89 and Ralpha145 in the alpha2-domain form an inter alpha-chain salt bridge between pairs of alphabeta-heterodimers. Other residues, Qalpha92 and Nalpha115, may be involved in close association in that part of the alpha-chain. We investigated the role of these amino acids on class II expression and antigen presentation. Class II composed of an Ealpha89K substituted alpha-chain paired with a wt beta-chain exhibited inhibited antigen presentation and expression of alpha-chain serologic epitopes. In contrast, mutation of Ralpha145E had less affect on antigen presentation and did not affect I-Ak serologic epitopes. Interchanging charges of the salt bridge residues by expressing both Ralpha145E and Ealpha89K on the same chain obviated the large negative effect of the Ealpha89K mutation on antigen presentation but not on the serologic epitopes. Our results are similar for those reported for mutation of DR3's inter-chain salt bridge with the exception that double mutants did not moderate the DR3 defect. Interestingly, the amino acids differences between I-A and DR change the location of the inter-chain salt bridges. In DR1 these residues are located at positions Ealpha88 and Kalpha111; in I-Ak these residues are located at position Ealpha89 and Ralpha145. Inter alpha-chain salt bridges are thus maintained in various class II molecules by amino acids located in different parts of the alpha2-domain. This conservation of structure suggests that considerable functional importance may attach to the ionic interactions.

Class II-targeted antigen is superior to CD40-targeted antigen at stimulating humoral responses in vivo.

We examined the efficacy of using monoclonal antibodies to target antigen (avidin) to different surface molecules expressed on antigen presenting cells (APC). In particular, we targeted CD40 to test whether the "adjuvant" properties of CD40 signaling combined with targeted antigen would result in enhanced serologic responses. We targeted avidin to class II as a positive control and to CD11c as a negative control. These surface proteins represent an ensemble of surface molecules that signal upon ligation and that are expressed on professional APC, in particular dendritic cells (DC). We observed that targeting class II molecules on APC was superior to targeting CD40, or CD11c. However, CD40 and CD11c could function as targets for antigen bound monoclonal antibodies under certain conditions. Interestingly, inclusion of anti-CD40 mAb with the targeting anti-class II-targeted antigens negatively affects humoral response, suggesting that CD40 signaling under certain conditions may suppress processing and/or presentation of targeted antigen.

Effects of infection with Enterobacteriaceae enteropathogens on subsequent infection with Ascaris suum in the laboratory mouse.

Prior to infection with Ascaris suum, ICR strain mice were inoculated with Salmonella typhisuis intraperitoneally or via gastric gavage. Similarly, Salmonella cholerae-suis var. kunzendorf, Salmonella typhimurium and enterotoxigenic Escherichia coli were administered to mice via gastric gavage 2 weeks prior to A. suum inoculation. Previous inoculation with S. typhisuis, via the intraperitoneal or gastric gavage routes and S. cholerae-suis var. kunzendorf decreased recovery of ascarid larvae from mice lungs. This effect appeared to be due to entrapment of migrating larvae by inflammatory reactions in the liver. This reaction was suspected to be due to non-specific resistance stimulated by the prior exposure to the bacterial pathogen. The number of A. suum larvae in the lungs of mice previously inoculated with S. typhimurium or enterotoxigenic E. coli (ETEC) was variable and in some cases greater in mice which had received the bacterial inoculation.

A tongue biopsy technique for the detection of trichinosis in swine.

A tongue biopsy technique developed for the detection of Trichinella spiralis infection in swine involves taking a deep core biopsy of the tongue musculature, and examination of the sample by digestion. Using this procedure, 31 of 52 (60%) swine from an Indiana herd were found to be infected with T. spiralis. The average biopsy weighed 0.42 g, and the intensity of infection averaged 180 larvae per gram (range 2-1157). The biopsy was quick and easy to perform and the tongues healed well following the procedure. This technique may have applicability for Trichinella detection in epidemiological, control and research studies on swine and other animals.

Immunochemical characterization of proteins from scrapie-infected hamster brain, using immunoblot analysis.

Preparations of brain plasma membrane from scrapie-infected or noninfected hamsters were extracted with a solvent and were used to inoculate rabbits. Antisera evaluated by immunoblot analysis revealed a protein of 45 kD in scrapie-infected hamster brain that had a greater signal compared with proteins of comparable relative mass in noninfected brain. This 45-kD protein was not increased in scrapie-infected mouse, sheep, or goat brain. Seemingly, the 45-kD protein may be a degradation product of glial fibrillary acidic protein.

Increased inhibition of proliferation of human B cell lymphomas following ligation of CD40, and either CD19, CD20, CD95 or surface immunoglobulin.

Non-Hodgkin's (NHL) B cell lymphomas are growth-inhibited by ligation of their CD40 molecules. This inhibition is not absolute in that approximately 50% of the cells are not inhibited. We conducted studies to see if other signals that have been reported to inhibit B cell lymphoma growth could be used in combination with anti-CD40 signaling to completely inhibit growth. Ligation of surface immunoglobulin (Ig), CD19, CD20, CD37 or CD95 with soluble antibody did not affect growth of the panel of NHL cells examined. Ligation of CD20, CD19 or CD95 was inhibitory for some NHL cell lines if the primary antibody was crosslinked with a secondary antibody. Combining anti-CD40 with anti-CD19, anti-CD20, or anti-Ig resulted in increased inhibition past that produced by anti-CD40 alone. The additive effect of anti-CD40 and other antibodies to selected surface markers was not observed in all NHL cell lines. Crosslinking of CD95 was also growth inhibitory for the majority of the NHL, and when combined with anti-CD40 under conditions that afforded crosslinking of the two receptors, increased inhibition was seen in three of the NHL cell lines. We found that cAMP or sodium butyrate (NaB) were also effective at inhibiting growth of the NHL cells; this was a profound inhibition (approaching 100%) compared to the 50% inhibition seen with anti-CD40 treatment. The potential for anti-CD40 and either cAMP or NaB to be additive was tested and not found to be the case. The ability to inhibit proliferation of the NHL was very dynamic with some antibody combinations being either inhibitory for multiple cells, not having an effect at all, or in some cases being stimulatory. This suggests that the NHL may represent unique stages of B cells that might serve as a model system which could be developed to precisely categorize patient NHL.

The ins and outs of getting in: structures and signals that enhance BCR or Fc receptor-mediated antigen presentation.

Antigen-presenting cells internalize antigen by fluid-phase pinocytosis or by endocytosis via surface receptors such as the B cell receptor (BCR) and Fc receptors for IgG, IgA and IgE (FcR). While both modes of internalization lead to antigen presentation it is recognized that receptor-mediated endocytosis greatly enhances the efficiency of processing and antigen presentation. Receptors facilitate the entry of antigen into the endocytic pathway by interaction of their internalization motifs with the endocytic machinery. These motifs include tyrosine-based, dileucine and casein kinase-like motifs. However these structures appear insufficient to support processing of cryptic epitopes, leading to a limited immune response. Cryptic epitope processing appears dependent on receptor signaling which is mediated by immunoreceptor tyrosine activation motifs (ITAMs). The signaling cascade which follows receptor crosslinking promotes reorganization and acidification of the late endocytic compartment or MIIC. Signaling events downstream of Syk, in particular calcium flux and protein kinase C activation, are necessary for MIIC induction. PI(3) kinase is also involved at multiple steps in antigen presentation, including production of PIP3 and transport of cathepsins. PIP3 is crucial both as a binding substrate for proteins implicated in vesicle transport and for the recruitment of signaling molecules to the plasma membrane. Among PIP3 activated molecules, protein kinase B (PKB) has been linked to endocytic function. We observe association of activated PKB with the MIIC after signaling through antigen presentation-competent receptors, but not mutant, presentation-defective receptors.

Anti-class II monoclonal antibody-targeted Vibrio cholerae TcpA pilin: modulation of serologic response, epitope specificity, and isotype.

Toxin-coregulated pilus (TCP) is a colonization factor required for cholera infection. It is not a strong immunogen when delivered in the context of whole cells, yet pilus subunits or TcpA derivative synthetic peptides induce protective responses. We examined the efficacy of immunizing mice with TCP conjugated to anti-class II monoclonal antibodies (MAb) with or without the addition of cholera toxin (CT) or anti-CD40 MAb to determine if the serologic response to TcpA could be manipulated. Anti-class II MAb-targeted TCP influenced the anti-TCP peptide serologic response with respect to titer and isotype. Responses to TcpA peptide 4 were induced with class II MAb-targeted TCP and not with nontargeted TCP. Class II MAb-targeting TcpA reduced the response to peptide 6 compared to the nontargeted TCP response. Class II MAb-targeted TcpA, if delivered with CT, enhanced the serologic response to TcpA peptides. The effectiveness of the combination of targeted TCP and CT was reduced if anti-CD40 MAb were included in the primary immunization. These data establish the need to understand the role of TCP presentation in the generation of B-cell epitopes in order to optimize TcpA-based cholera vaccines.

Evaluation of cholera vaccines formulated with toxin-coregulated pilin peptide plus polymer adjuvant in mice.

Cholera is an acute diarrheal disease that is caused by the gram-negative bacterium Vibrio cholerae. The low efficacy of currently available killed-whole-cell vaccines and the reactinogenicity coupled with potential reversion of live vaccines have thus far precluded widespread vaccination for the control of cholera. Recent studies on the molecular nature of the virulence components that contribute to V. cholerae pathogenesis have provided insights into possible approaches for the development of a defined subunit cholera vaccine. Genetic analysis has demonstrated that the toxin-coregulated pilus (TCP) is the major factor that contributes to colonization of the human intestine by V. cholerae. In addition, polyclonal and several monoclonal antibodies directed against TCP have been shown to provide passive immunity to disease in the infant mouse cholera model. In the present study, synthetic peptides corresponding to portions of the C-terminal disulfide region of TcpA pilin were formulated with polymer adjuvants currently in clinical trials and used to actively immunize adult female CD-1 mice. The experimental vaccine formulations elicited high levels of antigen-specific immunoglobulin G (IgG), including a broad spectrum of subclasses (IgG1, IgG2a, IgG2b, and IgG3), and lower levels of IgA. Infant mice born to the immunized mothers showed 100% protection against a 50% lethal dose (1 LD(50)) challenge and 50% protection against a 10-LD(50) challenge with virulent strain O395. These results indicate that specific regions of TcpA, including those delineated by the peptides used in this study, have the potential to be incorporated into an effective defined subunit vaccine for cholera.

Gamma-chain dependent recruitment of tyrosine kinases to membrane rafts by the human IgA receptor Fc alpha R.

We show that the human IgA receptor, Fc alpha R, redistributes to plasma membrane rafts after cross-linking and that tyrosine kinases are relocated to these sites following Fc alpha R capping. We demonstrate by confocal microscopy that Fc alpha R caps in membrane rafts by a gamma-chain-independent mechanism but that gamma-chain expression is necessary for Lyn redistribution. Immunoblotting of rafts isolated by sucrose density gradient centrifugation demonstrated recruitment of gamma-chain and phosphorylated tyrosine kinases Lyn and Bruton's tyrosine kinase to membrane rafts after Fc alpha R cross-linking. Time-dependent differences in Lyn phosphorylation and Bruton's tyrosine kinase distribution were observed between cells expressing Fc alpha R plus gamma-chain and cells expressing Fc alpha R only. This study defines early Fc alpha R-triggered membrane dynamics that take place before Fc alpha R internalization.

Characterization of nucleic acids in membrane vesicles from scrapie-infected hamster brain.

This study reports the partial characterization of nucleic acids present in gradient fractions enriched for large membrane vesicles from scrapie-infected and uninfected hamster brains. Labeling of phenol-extracted nucleic acids at the 3' or 5' ends revealed abundant amounts of low-molecular-weight RNA and little or no DNA. These nucleic acids survived nuclease treatment of membrane vesicles but were sensitive to RNase after phenol extraction. Analysis of 5'-end-labeled nucleic acids by one- and two-dimensional gel electrophoresis revealed an RNA of ca. 100 bases in preparations from scrapie-infected hamster brain that could not be detected in uninfected brain. The possibility that this apparently unique small RNA may result from tissue damage or abnormal RNA processing or may be a component of the infectious complex is discussed.