Communication between distal chromosomal elements is essential for control of many nuclear processes. For example, genes in higher eukaryotes often require distant enhancer sequences for high-level expression. The mechanisms proposed for long-range enhancer action fall into two basic categories. Non-contact models propose that enhancers act at a distance to create a favorable environment for gene transcription, or act as entry sites or nucleation points for factors that ultimately communicate with the gene. Contact models propose that communication occurs through direct interaction between the distant enhancer and the gene by various mechanisms that 'loop out' the intervening sequences. Although much attention has focused on contact models, the existence and nature of long-range interactions is still controversial and speculative, as there is no direct evidence that distant sequences physically interact in vivo. Here, we report the development of a widely applicable in situ technique to tag and recover chromatin in the immediate vicinity of an actively transcribed gene. We show that the classical enhancer element, HS2 of the prototypical locus control region (LCR) of the beta-globin gene cluster, is in close physical proximity to an actively transcribed HBB (beta-globin) gene located over 50 kb away in vivo, suggesting a direct regulatory interaction. The results give unprecedented insight into the in vivo structure of the LCR-gene interface and provide the first direct evidence of long-range enhancer communication.

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and by the G protein-coupled receptor agonist thrombin. The glycoprotein VI (GPVI)-specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.

Filamentous inclusions composed of the microtubule-associated protein tau are a defining characteristic of a large number of neurodegenerative diseases. Here we show that tau degradation in stably transfected and non-transfected SH-SY5Y cells is blocked by the irreversible proteasome inhibitor lactacystin. Further, we find that in vitro, natively unfolded tau can be directly processed by the 20S proteasome without a requirement for ubiquitylation, and that a highly reproducible pattern of degradation intermediates is readily detectable during this process. Analysis of these intermediates shows that 20S proteasomal processing of tau is bi-directional, proceeding from both N- and C-termini, and that populations of relatively stable intermediates arise probably because of less efficient digestion of the C-terminal repeat region. Our results are consistent with an in vivo role for the proteasome in tau degradation and support the existence of ubiquitin-independent pathways for the proteasomal degradation of unfolded proteins.

Mice lacking the p110delta catalytic subunit of phosphatidylinositol 3-kinase have reduced numbers of B1 and marginal zone B cells, reduced levels of serum immunoglobulins, respond poorly to immunization with type II thymus-independent antigen, and are defective in their primary and secondary responses to thymus-dependent antigen. p110delta(-/-) B cells proliferate poorly in response to B cell receptor (BCR) or CD40 signals in vitro, fail to activate protein kinase B, and are prone to apoptosis. p110delta function is required for BCR-mediated calcium flux, activation of phosphlipaseCgamma2, and Bruton's tyrosine kinase. Thus, p110delta plays a critical role in B cell homeostasis and function.

Sheep form an olfactory recognition memory for their lambs within 2 h of parturition and will subsequently reject the approaches of any strange lamb and protest vocally. In this study we report that following olfactory memory formation, ewes exposed to either their own or a strange lamb show c-fos mRNA expression in the medial frontal cortex, although levels of expression in the pyramidal output cell layer V were significantly higher in ewes that rejected strange lambs. Reversibly inactivating this region by the retrodialysis of the anaesthetic tetracaine before birth reduced aggressive motor responses towards lambs but not protest vocalisations. Similar treatment during the critical period for olfactory memory formation and lamb recognition (0-4 h post-partum) had no effect on ewes maternal behaviour towards their own lambs. It did, however, prevent the normal selective expression of aggressive rejection, and reduced protest vocalisation behaviours directed towards strange lambs. These rejection behaviours did appear 1 h after the termination of tetracaine infusions despite the ewes not being given the opportunity to interact with their own lambs during this time. Therefore, tetracaine blockade of the medial frontal cortex prevents animals from responding with motor aggression, but not vocal aggression, to odour cues from strange lambs, but has no effect on the formation of an olfactory recognition memory for their own lambs. Both pre- and post-partum aggressive rejection of strange lambs was associated with increased concentrations of dopamine, serotonin, glutamate and GABA. When these behaviours were inhibited by the tetracaine infusions, extracellular concentrations of these neurotransmitters were all increased by the anaesthetic but did not change in response to lambs. These findings suggest that a functional medial frontal cortex is not required for the formation of an olfactory recognition memory or for mediating pro-active maternal behaviours. It is however required for the mediation of motor but not vocal aspects of aggressive rejection responses directed towards aversive odour cues from strange lambs.

Metalloproteinases (MMP) produced by both cancer and normal stromal fibroblast cells play a critical role in the metastatic spread of tumours, however little is known of the regulation of their release. In this report we demonstrate that breast cancer cells in culture release apparently full length soluble EMMPRIN that promotes the release of pro-MMP2 from fibroblasts. The generation of MMP2 is mediated by activation of phospholipase A(2) and 5-lipoxygenase. These results suggest that the production of soluble EMMPRIN, phospholipase A(2) and 5-lipoxygenase activities are sites for potential therapeutic intervention.

In recent years, substantial progress has been made towards the identification of intracellular signalling molecules that couple multi-subunit immune-recognition receptors (MIRRs) to their various effector functions. Among these, the VAV proteins have been observed to have a crucial role in regulating some of the earliest events in receptor signalling. VAV proteins function, in part, as guanine-nucleotide exchange factors (GEFs) for the RHO/RAC family of GTPases. This review focuses on the role of VAV proteins in the regulation of lymphocyte development and function, and emphasizes the regulatory roles that these proteins have through both GEF-dependent and -independent mechanisms.

Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270 kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200 kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Le(a). After the release of oligosaccharides by reductive beta-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex(0-->2)NeuAc(1-->)(x)Hex(x)HexNAc(x)(-ol), x=1-6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Le(a) epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.

Rac, a member of the Rho family of monomeric GTPases, is an integrator of intracellular signaling in a wide range of cellular processes. We have purified a PtdIns(3,4,5)P3-sensitive activator of Rac from neutrophil cytosol. It is an abundant, 185 kDa guanine-nucleotide exchange factor (GEF), which we cloned and named P-Rex1. The recombinant enzyme has Rac-GEF activity that is directly, substantially, and synergistically activated by PtdIns(3,4,5)P3 and Gbetagammas both in vitro and in vivo. P-Rex1 antisense oligonucleotides reduced endogenous P-Rex1 expression and C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line. P-Rex1 appears to be a coincidence detector in PtdIns(3,4,5)P3 and Gbetagamma signaling pathways that is particularly adapted to function downstream of heterotrimeric G proteins in neutrophils.

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCalpha at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase-dependent stimulation of Rac1, ARF6, and PKCalpha.

We have analyzed the effects of deficiency in the tyrosine kinase Lyn on B cell development using transgenic mice that express a B cell antigen receptor (BCR) of defined specificity (3-83,anti-H-2K(k or b)). In the absence of Lyn, immature B cells are abundant in the bone marrow and spleen up until the T1 stage (IgM(hi) IgD(-) CD21(-)CD23(-)), after which B cell development is severely impaired. The small number of mature B cells that do develop in Lyn-deficient mice express normal levels of the transgenic BCR and lack expression of CD80 and CD86, suggesting they are not activated. In Lyn-deficient animals the presence of a Bcl-2 transgene leads to a dramatic increase in B cell numbers and restores T2 stage (IgM(hi) IgD(hi) CD21(hi) CD23(int)) and mature populations. In 3-83 lyn-/- Bcl-2 Tg mice, a population of lambda-positive cells that also express the 383 idiotype is evident, suggesting that in the absence of lyn isotype exclusion by the transgenic BCR is less efficient. The results indicate that Lyn plays a positive role in the selection and survival of mature B cells in addition to its previously documented negative role in tolerance and B cell activation.

The intracellular signals that trigger natural cytotoxicity have not been clearly determined. The Syk and ZAP-70 tyrosine kinases are essential for cellular activation initiated by B and T cell antigen receptors and may drive natural killer (NK) cell cytotoxicity via receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs). However, we found that, unlike B and T cells, NK cells developed in Syk-/-ZAP-70-/- mice and, despite their nonfunctional ITAMs, lysed various tumor targets in vitro and eliminated tumor cells in vivo, including those without NKG2D ligands. The simultaneous inhibition of phosphatidyl inositol 3 kinase and Src kinases abrogated the cytolytic activity of Syk-/-ZAP-70-/- NK cells and strongly reduced that of wild-type NK cells. This suggests that distinct and redundant signaling pathways act synergistically to trigger natural cytotoxicity.

Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.

Arabinogalactan proteins (AGPs) are proteoglycans secreted by plant cells that have been implicated in plant growth and development. Most AGPs cloned to date possess highly labile glycosylphosphatidylinositol (GPI) lipid anchors. These anchors transiently attach AGPs to the plasma membrane before they are released into the cell wall following GPI anchor hydrolysis. We have isolated and partially sequenced the protein core of an AGP purified from styles of Nicotiana alata. The protein sequence data were utilised to clone the AGP's gene, NaAGP4. This AGP shares about 78% sequence identity with the tomato AGP LeAGP-1. RNA gel blot analyses of different plant organs indicate that NaAGP4 is expressed in the same tissues and at similar levels as LeAGP-1. Furthermore, NaAGP4 like LeAGP-1 is rapidly suppressed by tissue wounding and by pathogen infection. We believe NaAGP4 and LeAGP-1 are the first described examples of orthologous AGPs from different plant species. In contrast, another AGP from N. alata, NaAGP1, is comparatively unaffected by wounding and pathogen infection, although this AGP is expressed in similar tissues and at similar levels as NaAGP4.

The human brain has evolved specialized neural mechanisms for visual recognition of faces, which afford us a remarkable ability to discriminate between, remember and think about many hundreds of different individuals. Sheep also recognize and are attracted to individual sheep and humans by their faces, as they possess similar specialized neural systems in the temporal and frontal lobes for assisting in this important social task, including a greater involvement of the right brain hemisphere. Here we show that individual sheep can remember 50 other different sheep faces for over 2 years, and that the specialized neural circuits involved maintain selective encoding of individual sheep and human faces even after long periods of separation.

B lymphocytes from mice null for the Rho-family guanine-nucleotide exchange factor, Vav, are defective in their ability to proliferate in response to BCR cross-linking, but are able to proliferate normally in response to LPS. In addition, they have a depletion of CD5(+) (B1) lymphocytes and defective IgG class switching. This phenotype is reminiscent of that observed in mice null for the cell cycle regulatory protein, cyclin D2. We demonstrate here that the inability of vav(-/-) B cells to proliferate in response to BCR ligation is due to an inability to induce cyclin D2. In addition, we show that the proliferative defect of these cells occurs after the cells have entered early G1 phase. Analyses of potential down-stream signaling intermediates revealed differential activation of the stress-activated MAP kinases in the absence of Vav, normal activation of the ERK, MAPK, and phosphatidylinositol 3-kinase pathways, and defective intracellular calcium mobilization. We further demonstrate that intracellular calcium homeostasis is required for cyclin D2 induction, implicating a possible link with the defective calcium response of vav(-/-) B cells and their inability to induce cyclin D2.

The extent to which "nurture" as opposed to "nature" determines behavior and sociosexual preferences in mammalian species is controversial although most recent interest has focused on genetic determinants. We report here that if sheep and goats are cross-fostered at birth, but raised in mixed-species groups, their play and grooming behavior resembles that of their foster rather than genetic species. There are no sex differences in effects on these behaviors, and other species-specific behavior patterns such as aggression, browsing, climbing, and vocalizations are unaffected. In adulthood, cross-fostered males strongly prefer to socialize and mate with females of their foster mother's species, even if raised with a conspecific of their own species. Castration within 2 days of birth slightly reduces the level of this altered social preference but mating preference following short-term testosterone treatment is the same as for gonadally intact animals. Cross-fostered females also show significant preference for socializing with females and mating with males of their foster mother's species, although this effect is weaker than that in both gonadally intact and castrated males. When cross-fostered animals are placed in flocks containing members of only their genetic species for 3 years, male social and mating preferences for females of their mother's species remain virtually unaffected. Females change to display an exclusive mating preference for members of their genetic species in 1-2 years although they still retain some social interest in female members of their foster species. Thus, there are clear sex differences in the impact of the emotional bond between a mother and her offspring in these mammals. Effects on males are strongest and irreversibly maintained even after altering their social environment, whereas those on females are weaker and mating preferences are clearly adaptable in the face of altered social priorities. These sex differences are presumably caused by pre-, or early postnatal, organizational effects of sex hormones on the brain.

Rat natural killer (NK) cells recognize MHC-I molecules encoded by both the classical (RT1-A) and non-classical (RT1-C/E/M) MHC class I (MHC-I) regions. We have identified a receptor, the STOK2 antigen, which belongs to the Ly-49 family of killer cell lectin-like receptors, and we have localized the gene encoding it to the rat natural killer cell gene complex. We have also shown that it inhibits NK cytotoxicity when recognizing its cognate MHC-I ligand RT1-A1c on a target cell. This is the first inhibitory Ly-49-MHC-I interaction identified in the rat and highlights the great similarity between rat and mouse Ly-49 receptors and their MHC ligands. However, the mode of rat NK-cell recognition of target cells indicates that positive recognition of allo-MHC determinants, especially those encoded by the RT1-C/E/M region, is a prevalent feature. NK cells recruited to the peritoneum as a consequence of alloimmunization display positive recognition of allodeterminants. In one case, NK cells activated in this way have been shown to be specific for the immunizing, non-classical class I molecule RT1-Eu. These findings show that allospecific NK cells sometimes show features reminiscent of the adaptive immune response.

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.