We describe a format for production of protein arrays termed 'protein in situ array' (PISA). A PISA is rapidly generated in one step directly from PCR-generated DNA fragments by cell-free protein expression and in situ immobilisation at a surface. The template for expression is DNA encoding individual proteins or domains, which is produced by PCR using primers designed from information in DNA databases. Coupled transcription and translation is carried out on a surface to which the tagged protein adheres as soon as it is synthesised. Because proteins generated by cell-free synthesis are usually soluble and functional, this method can overcome problems of insolubility or degradation associated with bacterial expression of recombinant proteins. Moreover, the use of PCR-generated DNA enables rapid production of proteins or domains based on genome information alone and will be particularly useful where cloned material is not available. Here we show that human single-chain antibody fragments (three domain, V(H)/K form) and an enzyme (luciferase) can be functionally arrayed by the PISA method.

The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b(558) (gp91(phox) and p22(phox)) and three soluble factors (GTP-Rac, p47(phox) and p67(phox) (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40(phox) has subsequently been discovered as a binding partner for p67(phox) (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation but through an unknown mechanism. We show that the addition of p40(phox) to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns(3)P to the PX domain of p40(phox). These results offer new insights into the roles for PI(3)Ks and p40(phox) in ROS formation and define a cellular ligand for the orphan PX domain.

Increasing evidence suggests that the effect of HLA-E on Natural Killer (NK) cell activity can be affected by the nature of the peptides bound to this non-classical, MHC class Ib molecule. However, its reduced cell surface expression, and until recently, the lack of specific monoclonal antibodies hinder studying the peptide-binding specificity HLA-E.

The product of the protooncogene Vav1 participates in multiple signaling pathways and is a critical regulator of antigen-receptor signaling in B and T lymphocytes, but its role during in vivo natural killer (NK) cell differentiation is not known. Here we have studied NK cell development in Vav1-/- mice and found that, in contrast to T and NK-T cells, the absolute numbers of phenotypically mature NK cells were not reduced. Vav1-/- mice produced normal amounts of interferon (IFN)-gamma in response to Listeria monocytogenes and controlled early infection but showed reduced tumor clearance in vivo. In vitro stimulation of surface receptors in Vav1-/- NK cells resulted in normal IFN-gamma production but reduced tumor cell lysis. Vav1 was found to control activation of extracellular signal-regulated kinases and exocytosis of cytotoxic granules. In contrast, conjugate formation appeared to be only mildly affected, and calcium mobilization was normal in Vav1-/- NK cells. These results highlight fundamental differences between proximal signaling events in T and NK cells and suggest a functional dichotomy for Vav1 in NK cells: a role in cytotoxicity but not for IFN-gamma production.

B and T lymphocytes develop normally in mice lacking the guanine nucleotide exchange factor Vav-2. However, the immune responses to type II thymus-independent antigen as well as the primary response to thymus-dependent (TD) antigen are defective. Vav-2-deficient mice are also defective in their ability to switch immunoglobulin class, form germinal centers and generate secondary immune responses to TD antigens. Mice lacking both Vav-1 and Vav-2 contain reduced numbers of B lymphocytes and display a maturational block in the development of mature B cells. B cells from Vav-1(-/-)Vav-2(-/-) mice respond poorly to antigen receptor triggering, both in terms of proliferation and calcium release. These studies show the importance of Vav-2 in humoral immune responses and B cell maturation.

During the assembly of major histocompatibility complex (MHC) class I molecules transient associations are formed with the endoplasmic reticulum resident chaperones calnexin and calreticulin, ERp57 oxidoreductase, and also with tapasin, the latter mediating binding of the class I molecules to the transporter associated with antigen processing (TAP). We report here the isolation of a cDNA encoding rat tapasin from a DA (RT1av1) library. The cDNA encodes a proline-rich (11.3%) polypeptide of 464 residues with a potential ER-retention KK motif at its COOH-terminus, and a predicted molecular mass of 48 kDa. Matrix-assisted laser-desorption ionisation (MALDI) mass spectrometry of peptides derived from in-gel tryptic digestion of a TAP-associated protein match regions of the predicted translation product. A species of the correct molecular mass and predicted pl was also identified in association with radiolabelled immunoprecipitates of the rat TAP complex analysed by two-dimensional gel electrophoresis. This confirms rat tapasin as a component of the rat MHC class I assembly complex.

The rat MHC class Ia molecule RT1-Aa has the unusual capacity to bind long peptides ending in arginine, such as MTF-E, a thirteen-residue, maternally transmitted minor histocompatibility antigen. The antigenic structure of MTF-E was unpredictable due to its extraordinary length and two arginines that could serve as potential anchor residues. The crystal structure of RT1-Aa-MTF-E at 2.55 A shows that both peptide termini are anchored, as in other class I molecules, but the central residues in two independent pMHC complexes adopt completely different bulged conformations based on local environment. The MTF-E epitope is fully exposed within the putative T cell receptor (TCR) footprint. The flexibility demonstrated by the MTF-E structures illustrates how different TCRs may be raised against chemically identical, but structurally dissimilar, pMHC complexes.

Phosphatidylinositol 3-kinases are a family of dual specificity lipid/protein kinases. The products of PI3K's, phosphatidylinositol(3,4,5) triphosphate and phosphatidylinositol(3,4) bisphosphate, act as second messengers connecting activated transmembrane receptors to signaling pathways that control gene transcription, proliferation, transformation, programmed cell death, adhesion, migration and vesicular transport. There is evidence that different isoforms of PI3K's activate specific signaling pathways and are thus responsible for integrating cellular responses. The elucidation of the genomic structure of the catalytic subunits is a necessary step for the investigation of the function of PI3K isoforms by inactivation of the gene in vivo. The structural organization of p110alpha, beta, and gamma genes has been previously reported. Here we report the cloning, sequencing, and structural organization of the mouse p110delta gene from a murine 129/Sv genomic library. The p110delta gene consists of 22 exons and spans over 13 kb. Comparison of the genomic structure with that of p110alpha, beta, and gamma demonstrates that the p110delta gene shares its exon structure with p110beta, the most closely related PI3K at the amino acid level.

The cDNA of a type 1 ADP-ribosylation factor (ARF) from the desert locust, Locusta migratoria was cloned, sequenced and compared to ARF1 genes of other species. The locust ARF1 protein is 100% identical with the ARF1 protein of the fruit fly Drosophila melanogaster even though the DNA sequences are only 79% identical. The significance of this finding in relation to the considerable evolutionary distance between hemimetabolous and holometabolous insects is discussed.

We show here that Vav-2 is tyrosine phosphorylated following antigen receptor engagement in both B- and T-cells, but potentiates nuclear factor of activated T cells (NFAT)-dependent transcription only in B cells. Vav-2 function requires the N-terminus, as well as functional Dbl homology and SH2 domains. More over, the enhancement of NFAT-dependent transcription by Vav-2 can be inhibited by a number of dominant-negative GTPases. The ability of Vav-2 to potentiate NFAT-dependent transcription correlates with its ability to promote a sustained calcium flux. Thus, Vav-2 augments the calcium signal in B cells but not T cells, and a truncated form of Vav-2 can neither activate NFAT nor augment calcium signaling. The CD19 co-receptor physically interacts with Vav-2 and synergistically enhances Vav-2 phosphorylation induced by the B-cell receptor (BCR). In addition, we found that Vav-2 augments CD19-stimulated NFAT- dependent transcription, as well as transcription from the CD5 enhancer. These data suggest a role for Vav-2 in transducing BCR signals to the transcription factor NFAT and implicate Vav-2 in the integration of BCR and CD19 signaling.

The agonist-specific coupling properties of the three cloned human alpha(2)-adrenoceptor subtypes have been compared, when expressed at similar levels in Chinese hamster ovary (CHO) cell lines, using noradrenaline and (+/-)-meta-octopamine as agonists. Noradrenaline can couple the receptor to both the inhibition and stimulation of forskolin-stimulated cyclic AMP production in all three receptor subtypes, with the relative strength of the coupling to the pathways varying for each of the receptor subtypes. meta-Octopamine selectively couples the alpha(2A)-adrenoceptor only to the inhibition of forskolin-stimulated cyclic AMP production. However, meta-octopamine couples the alpha(2B)- and alpha(2C)-adrenoceptors to both the inhibition and stimulation of forskolin-stimulated cyclic AMP production. The relative potency of meta-octopamine to noradrenaline varies between the different alpha(2)-adrenoceptor subtypes. The effects of meta-octopamine are around two orders of magnitude less potent than those of noradrenaline on both the alpha(2A)- and alpha(2B)-adrenoceptor subtypes. In contrast, in the case of the alpha(2C)-adrenoceptor, meta-octopamine is only one order of magnitude less potent than noradrenaline in the stimulation of forskolin-stimulated cyclic AMP production and, in addition, is equipotent with noradrenaline in the inhibition of forskolin-stimulated cyclic AMP production and has an increased maximal response. This raises the possibility that meta-octopamine may have physiologically important actions via alpha(2C)-adrenoceptors in vivo. The results show that the modulation of cyclic AMP production occurs in both a subtype- and agonist-specific manner for alpha(2A)-adrenoceptors and in a subtype specific manner for alpha(2B)- and alpha(2C)-adrenoceptors.

The CD45 tyrosine phosphatase lowers T-cell antigen receptor signalling thresholds by its positive actions on p56(lck) tyrosine kinase function. We now show that mice expressing active lck(F505) at non-oncogenic levels develop aggressive thymic lymphomas on a CD45(-/-) background. CD45 suppresses the tumorigenic potential of the kinase by dephosphorylation of the Tyr394 autophosphorylation site. In CD45(-/-) thymocytes the kinase is switched to a hyperactive oncogenic state, resulting in increased resistance to apoptosis. Transformation occurs in early CD4(-)CD8(-) thymocytes during the process of TCR-beta chain rearrangement by a recombinase-independent mechanism. Our findings represent the first example in which a tyrosine phosphatase in situ prevents the oncogenic actions of a SRC: family tyrosine kinase.

The rat major histocompatibility complex class Ia allelomorph RT1-A1(c) is a potent ligand for the recently identified inhibitory rLy-49 receptor, STOK-2. With the ultimate objective of studying the interactions of these molecules using structural and functional methods, we undertook a detailed study of its peptide specificity. The study revealed that designing an "ideal peptide" by choosing the most abundant residues in the "binding motif" obtained by pool sequencing does not necessarily yield an optimal binding peptide. For RT1-A1(c), as many as four positions, P2, P4, P5, and P9, were detected as putative anchors. Since this molecule displays a preference for highly hydrophobic peptides, we tested binding of peptides derived from the known leader peptide sequences of other rat histocompatibility complex class I molecules. One such peptide, found to bind well, requiring 1.6 microm peptide to achieve 50% stabilization, was searched for in vivo. Natural RT1-A1(c) binding peptides were purified from rat splenocytes and characterized by mass spectrometry using a combined matrix-assisted laser desorption ionization/time-of-flight and quadrupole time-of-flight approach. Results showed that the signal sequence-derived peptide was not detectable in the purified peptide pool, which was composed of a complex spectrum of peptides. Seven of these self-peptides were successfully sequenced.

We have previously shown activation of NK cells via recognition of an allogeneic, non-classical MHC class I molecule, RT1-E(u). In this study we investigated whether a self-MHC class I molecule could protect the allogeneic targets from being recognized and killed by the alloreactive NK (allo NK) cells. NK cells from BN (RT1 n) rats, primed in vivo by immunization with RT1(u)-expressing cells, manifested cytolytic activity against RT1(u)- as well as RT1(u/lv1)-expressing targets, but not against RT1(u/n)-expressing targets. The absence of cytolytic activity against semiallogeneic targets, i.e. targets expressing self-allotypes, was also valid for allo NK cells from alloimmunized F344 (RT1 (lv1)) rats. To analyze the ability of a distinct MHC class I molecule to protect target cells from NK lysis, Rat2 cells transfected with the activating allogeneic MHC class Ib, RT1-E(u) molecule were also transfected with the self-MHC class Ia, RT1-A1(n) molecule. The allo NK cells from BN rats immunized with RT1(u)-expressing cells were cytolytic against Rat2 transfected with the RT1-E(u) molecule. However, the allo NK cells manifested no cytolytic activity against double-transfected Rat2 cells, expressing the RT1-E(u) as well as the RT1-A1(n) molecule. We conclude that expression of a self-MHC class Ia (RT1-A) molecule protects targets from allo NK killing. Furthermore, the NK inhibition via recognition of the self-MHC class Ia molecule dominates over the activation via recognition of the allogeneic MHC class Ib molecule, RT1-E.

A panel of 11 rat monoclonal antibodies (mAbs) has been raised to ovine placental lactogen (PL). By competitive enzyme-linked immunoabsorbent assay (ELISA), confirmed by two-site ELISA, the antibodies were shown to recognize six antigenic determinants on the ovine PL molecule, two of which overlap. One antigenic determinant (designated 1) was shared by other members of the prolactin/growth hormone (GH)/PL family in ruminants, humans and rodents. The binding of (125)I-labelled ovine PL to crude receptor preparations from sheep liver (somatotrophic) or rabbit mammary gland (lactogenic) was inhibited by mAbs recognizing antigenic determinants 2-6. Both types of receptor preparation were affected similarly. In the local in vivo pigeon crop sac assay, mAbs directed against determinants 3 and 6 enhanced the biological activity of ovine PL.

The Syk protein tyrosine kinase (PTK) is essential for B, but not T or NK, cell development, although certain T cell subsets (i.e., gamma delta T cells of intestine and skin) appear to be dependent on Syk. In this report, we have re-evaluated the role of Syk in T cell development in hematopoietic chimeras generated by using Syk-deficient fetal liver hematopoietic stem cells (FL-HSC). We found that Syk-/- FL-HSC were vastly inferior to wild-type FL-HSC in reconstituting T cell development in recombinant-activating gene 2 (RAG2)-deficient mice, identifying an unexpected and nonredundant role for Syk in this process. This novel function of Syk in T cell development was mapped to the CD44-CD25+ stage. According to previous reports, development of intestinal gamma delta T cells was arrested in Syk-/- -->RAG2-/- chimeras. In striking contrast, when hosts were the newly established alymphoid RAG2 x common cytokine receptor gamma-chain (RAG2/gamma c) mice, Syk-/- chimeras developed intestinal gamma delta T cells as well as other T cell subsets (including alpha beta T cells, NK1.1+ alpha beta T cells, and splenic and thymic gamma delta T cells). However, all Syk-deficient T cell subsets were reduced in number, reaching about 25-50% of controls. These results attest to the utility of chimeric mice generated in a low competitive hematopoietic environment to evaluate more accurately the impact of lethal mutations on lymphoid development. Furthermore, they suggest that Syk intervenes in early T cell development independently of ZAP-70, and demonstrate that Syk is not essential for the intestinal gamma delta T cell lineage to develop.

The complete nucleotide sequence of Tn10 has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis. The glutamate permease was fully functional when expressed, but Tn10 did not protect Escherichia coli from the toxic effects of various metals.

The tyrosine kinase SYK plays critical roles in signalling through immune receptors. Gene-targeting studies have identified the cell types that require SYK for development and function, and the receptors that use SYK as well as their downstream signalling effectors. There is also evidence of a role for SYK in non-immune cells and in the maintenance of vascular integrity.

Using random peptide libraries we have previously shown that both mouse and rat class I molecules can exhibit different peptide length preferences. Such studies required expression of the particular class I molecules in RMA-S, a cell line deficient in the transporter associated with antigen presentation (TAP). For another rat class I molecule called RT1-A(u), however, we found that expression in RMA-S was poor and could not be increased sufficiently by incubation at 26 degrees C. To circumvent this problem we performed our studies on C58, a rat cell line that expresses RT1-A(u) naturally in the presence of a functional TAP transporter. Using C58 cells, cell-surface-expressed class I molecules were 'stripped' of peptides and beta(2)-microglobulin by washing the cells with an acidic citrate buffer (pH 3.3). Peptide stabilization assays, assessed by FACS analysis, were then performed using either specific peptides or synthetic random peptide libraries of different lengths (7-15 amino acids), supplemented with recombinant rat beta(2)-microglobulin. As a positive control an RT1-A(u)-specific nonamer peptide was designed using the previously determined peptide binding motif and this was found to bind to RT1-A(u) at nanomolar concentrations. Both length preference and importance of free N- and C-termini were tested using free base, formylated and acetylated peptide libraries. Results showed that RT1-A(u) was not able to accommodate N- or C-terminally blocked peptides but displayed a preference for peptides of 9-12 amino acids, similar to the preference observed for the RT1-A1(c) allotype, the other rat TAP-B-associated molecule tested thus far. These results suggest that length preference remains a consideration to explain the allelic class I-TAP associations of the RT1-A region.

Arabinogalactan proteins (AGPs) are proteoglycans of higher plants, which are implicated in growth and development. We recently have shown that two AGPs, NaAGP1 (from Nicotiana alata styles) and PcAGP1 (from Pyrus communis cell suspension culture), are modified by the addition of a glycosylphosphatidylinositol (GPI) anchor. However, paradoxically, both AGPs were buffer soluble rather than membrane associated. We now show that pear suspension cultured cells also contain membrane-bound GPI-anchored AGPs. This GPI anchor has the minimal core oligosaccharide structure, D-Manalpha(1-2)-D-Manalpha(1-6)-D-Manalpha(1-4)-D-GlcN -inositol, which is consistent with those found in animals, protozoa, and yeast, but with a partial beta(1-4)-galactosyl substitution of the 6-linked Man residue, and has a phosphoceramide lipid composed primarily of phytosphingosine and tetracosanoic acid. The secreted form of PcAGP1 contains a truncated GPI lacking the phosphoceramide moiety, suggesting that it is released from the membrane by the action of a phospholipase D. The implications of these findings are discussed in relation to the potential mechanisms by which GPI-anchored AGPs may be involved in signal transduction pathways.

Previous studies have established that NK cells express both inhibitory and activatory receptors. The inhibitory receptors have been shown to recognize major MHC class I molecules, but the physiological ligands for the activatory receptors have been only partly characterized. In this study we investigated whether NK cells could be activated by recognizing specific non-classical MHC class Ib molecules. NK cells from BN (RT1(n)) rats immunized in vivo with MHC-incompatible WF (RT1(u)) cells displayed cytolytic activity specific for product(s) of the MHC class Ib RT1-E(u) / C(u) region. These cells were shown to kill Rat2 fibroblast cells transfected with cDNA for RT1-E(u) but neither untransfected Rat2 nor a transfectant with the class Ia allele, RT1-A(u). Cytolysis of Rat2-RT1-E(u) was inhibited by the anti-RT1-E(u) antibody 70-3-C2. In addition, NK cells cytolytic against PVG (RT1(c)) targets, but not against WF (RT1(u)) or other allogeneic targets were activated after PVG immunization of BN rats. The generation of NK populations cytolytic for target cells of the same haplotype as the immunizing cells, but not for third-party targets, strongly suggests the existence of a selective NK-mediated response in vivo. We conclude that recognition of an allogeneic MHC class Ib RT1-E molecule activates NK cells and the specific cytolytic response could be regarded as adaptive.

NK lymphocytes lyse certain xenogeneic cells without prior sensitization. The receptors by which NK cells recognize xenogeneic targets are largely uncharacterized but have been postulated to possess broad specificity against ubiquitous target ligands. However, previous studies suggest that mouse NK cells recognize xenogeneic targets in a strain-specific manner, implicating finely tuned, complex receptor systems in NK xenorecognition. We speculated that mouse Ly-49D, an activating NK receptor for the MHC I ligand, H2-Dd, might display public specificities for xenogeneic target structures. To test this hypothesis, we examined the lysis of xenogeneic targets by mouse Ly-49D transfectants of the rat NK cell line RNK-16 (RNK. Ly-49D). Of the xenogeneic tumor targets tested, RNK.Ly-49D, but not untransfected RNK-16, preferentially lysed tumor cells derived from Chinese hamsters and lymphoblast targets from rats. Ly-49D-dependent recognition of Chinese hamster cells was independent of target N-linked glycosylation. Mouse Ly-49D also specifically stimulated the natural killing of lymphoblast targets derived from wild-type and MHC-congenic rats of the RT1lv1 and RT1l haplotypes, but not of the RT1c, RT1u, RT1av1, or RT1n haplotypes. These studies demonstrate that Ly-49D can specifically mediate cytotoxicity against xenogeneic cells, and they suggest that Ly-49D may recognize xenogeneic MHC-encoded ligands.

The T cell repertoire is shaped by positive and negative selection of thymocytes. TCR-mediated signals that determine these selection processes are only partly understood. The CD45 tyrosine phosphatase has been shown to be important for signal transduction through the TCR, but there has been disagreement about whether CD45 is a positive or negative regulator of TCR signaling. Using CD45-deficient mice expressing transgenic TCR, we show that in the absence of CD45 there is a large increase in the thresholds of TCR stimulation required for both positive and negative selection. Our results conclusively demonstrate that in double-positive thymocytes CD45 is a positive regulator of the TCR signals that drive thymic selection events.

The pre-TCR complex regulates the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP) thymocytes during T cell development. In CD45(-/-) mice there is an accumulation of DN cells, suggesting a possible role for CD45 in pre-TCR signaling. We therefore crossed CD45(-/-) with Rag-1(-/-) mice to investigate the signaling functions of the CD3 complex in DN thymocytes. Remarkably, treatment of Rag-1(-/-)/CD45(-/-) mice with a CD3 mAb caused maturation to the DP stage at only 3% of the level measured in Rag-1(-/-) mice. Furthermore, ligation of the CD3 complex on Rag-1(-/-) /CD45(-/-) thymocytes in vitro induced less tyrosine phosphorylation in specific proteins when compared to Rag-1(-/-) thymocytes. CD45(-/-) mice were also crossed with pLGFA mice expressing a constitutively active form of the lck tyrosine kinase which restored the DN to DP transition to near normal levels. Our results are consistent with a model in which CD45-activated p56(lck) is critical for pre-TCR signal transduction.