С����Ƶ

Specific binding of spermatozoa to the zona pellucida that surrounds mammalian eggs is a key step in the fertilization process. However, the sperm proteins that recognise zona pellucida receptors remain contentious despite longstanding research efforts to identify them. Here we present evidence that proacrosin, a tissue-specific protein found within the acrosomal vesicle of all mammalian spermatozoa, is a multifunctional protein that mediates binding of acrosome-reacted spermatozoa to zona glycoproteins via a stereospecific polysulfate recognition mechanism. Using sulfated versus non-sulfated forms of chemically defined compounds in binding assays employing native proteins in their normal cellular location or conjugated to FluoSpheres, we have attempted to identify the sulfation "code" required for recognition. Results show that protein conformation is important for specificity and that at least 2 sulfate groups are required to cross-link spatially separated docking sites on proacrosin. The consistently most effective inhibitory compounds were suramin and quercetin-3beta-d-glucoside sulfate. The results support our hypothesis that proacrosin is one of several proteins in the acrosomal matrix that retain acrosome reacted spermatozoa on the zona surface prior to penetration. They also establish, as a proof-of-principle, the feasibility of synthesising sulfated compounds of high specificity as antifertility agents for human or animal use.

T-cell-receptor triggering and the delivery of co-stimulation are essential events leading to T cell expansion, differentiation and effector function. The influence that such signals exert on T cell migration during and following priming has been highlighted by recent reports. Moreover, induction of peripheral tolerance might act in part by affecting T cell migration. Here, we propose that the integration of co-stimulatory signals, which regulate the ability of primed T cells to access nonlymphoid tissue, and cognate recognition of the endothelium, which determines the selective recruitment of specific T cells, contribute to the anatomy of T cell-mediated immunity and tolerance. The implications for therapeutic strategies manipulating these signals are discussed.

MicroRNAs are a class of small RNAs that are increasingly being recognized as important regulators of gene expression. Although hundreds of microRNAs are present in the mammalian genome, genetic studies addressing their physiological roles are at an early stage. We have shown that mice deficient for bic/microRNA-155 are immunodeficient and display increased lung airway remodeling. We demonstrate a requirement of bic/microRNA-155 for the function of B and T lymphocytes and dendritic cells. Transcriptome analysis of bic/microRNA-155-deficient CD4+ T cells identified a wide spectrum of microRNA-155-regulated genes, including cytokines, chemokines, and transcription factors. Our work suggests that bic/microRNA-155 plays a key role in the homeostasis and function of the immune system.

In mammals, paternal and maternal pronuclei undergo profound chromatin reorganisation upon fertilisation. How these events are orchestrated within centromeric regions to ensure proper chromosome segregation in the following cellular divisions is unknown. In this study, we followed the dynamic unfolding of the centromeric regions, i.e. the centric and pericentric satellite repeats, by DNA fluorescent in situ hybridization (FISH) during the first cell cycle up to the two-cell stage. The distinct chromatin from female and male gametes both undergo rapid remodelling and reach a zygotic organisation in which the satellites occupy restricted spatial domains surrounding the nucleolar precursor body. A transition from this zygotic to a somatic cell-like organisation takes place during the two-cell stage. Using 3D immuno-FISH, we find that, whereas maternal pericentric regions are marked with H3K9me3, H4K20me3 and HP1beta, paternal ones only showed HP1beta marking. Thus, despite different chromatin features, male and female pronuclei organise their centromeric regions in the same way within the nuclei to align chromosomes on the metaphase plate and segregate them appropriately. Our findings highlight the importance of ensuring a proper centromere function while preserving the distinction of parental genome origin during the return to totipotency in the zygote.

The vesicular monoamine transporter type 2 (VMAT2) packages pre-synaptic monoamines into vesicles. Previously, we generated mice hypomorphic for the VMAT2 gene (Slc18a2), which results in a approximately 95% reduction in VMAT2 protein, disrupted vesicular storage, severe depletion of striatal dopamine and mice with moderate motor behaviour deficits. Dopamine released from mid-brain dopamine neurons acts on post-synaptic type 1 (D1) and 2 (D2) receptors located on striatal medium spiny neurons to initiate a signalling cascade that leads to altered transcription factor activity, gene expression and neuronal activity. We investigated striatal gene expression changes in VMAT2hypo mice by quantitative real-time PCR and in situ hybridisation. Despite unaltered expression of D1 and D2 dopamine receptors, there were dramatic alterations in striatal mRNAs encoding the neuropeptides substance P, dynorphin, enkephalin and cholecystokinin. The promoters of these genes are regulated by a combination of transcription factors that includes cAMP responsive element binding protein-1 (CREB) and c-Fos. Indeed, the changes in peptide mRNAs were associated with elevated expression of Creb1 and c-Fos. These data indicate that striatal dopamine depletion, as a consequence of deficient vesicular storage in this mouse, triggers a complex program of gene expression, consistent with this mouse being an excellent model of Parkinson's disease.

IL-17-producing T cells have recently been classified as a new effector T-cell subset, termed Th17, which is distinct from Th1, Th2 and Treg subsets. There has been much progress in the past year, leading to identification of the molecular mechanisms that drive differentiation of Th17 T cells. This has helped to clarify many aspects of their role in host defense as well as in autoimmunity. Nevertheless, many intriguing questions remain to be answered regarding the regulation of Th17-mediated responses as well as their interactions with the other T-cell subsets. Furthermore, the role of pathogens and pathogen-derived molecules in influencing effector T-cell polarization needs to be re-evaluated in the light of the differentiation conditions that favor Th17 T-cell responses.

L-selectin mediates tethering and rolling of lymphocytes in high endothelial venules (HEV) of lymph nodes (LN) and of leukocytes at inflammatory sites. We used transgenic mice expressing varying levels of wild-type or a non-cleavable mutant form of L-selectin on T cells to determine the relationship between L-selectin density, tethering and rolling, and migration into LN. T cells expressing supraphysiological levels of either wild-type or non-cleavable L-selectin showed rolling parameters similar to C57BL/6 T cells in hydrodynamic flow assays and during rolling in Peyer's patch HEV. In contrast, PMA- or antigen-activated T cells and L-selectin(+/-) T cells expressing subphysiological levels of L-selectin showed reduced numbers of rolling cells with increased rolling velocity. Short-term homing studies showed that elevated expression of L-selectin above physiological levels had no effect on T cell migration to LN; however, low L-selectin expression resulted in reduced T cell homing to LN. Thus, T lymphocyte migration into LN is regulated by the density of cell surface L-selectin. In addition, there is a saturable density of L-selectin required for optimal homing to PLN in C57BL/6 mice, the L-selectin level on circulating naive T cells promotes optimal homing, and increased expression above saturating levels promotes no further increase in T cell recruitment.

Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 A resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close to the C terminus, and each epitope comprises residues from both Cgamma3 domains. A central role in the unusually hydrophilic epitope is played by the side-chain of Arg355, accounting for the subclass specificity of RF61, which recognizes IgG1,-2, and -3 in preference to IgG4, in which the corresponding residue is Gln355. Compared with a previously determined complex of a lower affinity RF (RF-AN) bound to IgG4 Fc, in which only residues at the very edge of the antibody combining site were involved in binding, the epitope bound by RF61 is centered in classic fashion on the axis of the V(H):V(L) beta-barrel. The complementarity determining region-H3 loop plays a key role, forming a pocket in which Arg355 is bound by two salt-bridges. The antibody contacts also involve two somatically mutated V(H) residues, reinforcing the suggestion of a process of antigen-driven maturation and selection for IgG Fc during the generation of this RF autoantibody.

Genetic variants of interleukin 2 (IL-2) and its receptor are associated with murine and human susceptibility to Type 1 diabetes, yet the role of IL-2 in controlling pancreatic islet-reactive T cells is unknown. Here, we develop a model where IL-2 deficiency precipitates a breakdown of self-tolerance and progression to diabetes, and its action upon diabetogenic islet-specific CD4 T cells can be tracked. We find that IL-2 is not required for Aire-dependent thymic clonal deletion of high-avidity diabetogenic clones, but is essential for thymic formation of islet-specific Foxp3-expressing CD4 T cells. The absence of IL-2 results in the expansion of low-avidity Foxp3(-) islet-reactive CD4 T cells. The mechanism by which IL-2 prevents diabetes is therefore through the establishment of a repertoire of islet-reactive Foxp3(+) T cells within the thymus, and limitation of the peripheral activation of low-avidity islet-reactive T cells that normally escape thymic negative selection.

NK cell (natural killer cells) are lymphocytes of innate immunity that kill tumour cells and respond to infections, without prior stimulation. A balance of activating and inhibitory signals regulates NK cell cytotoxicity, but the molecular mechanisms are not fully understood. General inhibitors of PI3K (phosphoinositide 3-kinase) suppress cytotoxicity in human and mouse NK cells. However, which isoforms and how they regulate NK cell activation is unknown, and no data have been published on mice carrying PI3K mutations. p110delta expression is restricted to leucocytes, where it plays central roles in lymphocyte development and signalling. We have used mice carrying a catalytically inactive mutant form of p110delta in order to determine its role in NK cell biology. We show here that p110delta is not required to kill tumour cells, but unexpectedly p110delta mutant mice failed to fully reject transplanted lymphomas. Our results show for the first time a critical role for p110delta in NK cell biology in vivo.

PI3Ks (phosphoinositide 3-kinases) regulate diverse cellular functions such as metabolism, growth, gene expression and migration. The p110delta isoform of PI3K is mainly expressed in cells of the immune system and contributes to cellular and humoral immunity. In the thymus, p110delta and p110gamma play complementary roles in regulating the transition through key developmental checkpoints. In addition, p110delta regulates the differentiation of peripheral Th (helper T-cells) towards the Th1 and Th2 lineages. Moreover, p110delta is critical for Treg (regulatory T-cell) function. Here, we review the role of PI3Ks in T-cell development and function.

Pluripotent stem cells, similar to more restricted stem cells, are able to both self-renew and generate differentiated progeny. Although this dual functionality has been much studied, the search for molecular signatures of 'stemness' and pluripotency is only now beginning to gather momentum. While the focus of much of this work has been on the transcriptional features of embryonic stem cells, recent studies have indicated the importance of unique epigenetic profiles that keep key developmental genes 'poised' in a repressed but activatable state. Determining how these epigenetic features relate to the transcriptional signatures of ES cells, and whether they are also important in other types of stem cell, is a key challenge for the future.

Patients with certain forms of systematic vasculitis, such as Wegener's granulomatosis, have circulating antineutrophil cytoplasmic antibodies (ANCA). These inappropriately stimulate circulating neutrophils adhere to and thereby obstruct small vessels. This, together with ANCA-induced degranulation and an oxidative burst, leads to local tissue damage. The signaling pathways that are activated by ANCA IgG are distinct from those that are involved in normal neutrophil activation. This study shows that diacylglycerol kinase is selectively activated by ANCA and that the generated phosphatidic acid is responsible for promoting neutrophil adhesion, in part through integrin activation. The data presented point to diacylglycerol kinase alpha as a novel but selective target for the development of drugs to treat this potentially fatal disorder.

Foxp3 is essential for the commitment of differentiating thymocytes to the regulatory CD4(+) T (T reg) cell lineage. In humans and mice with a genetic Foxp3 deficiency, absence of this critical T reg cell population was suggested to be responsible for the severe autoimmune lesions. Recently, it has been proposed that in addition to T reg cells, Foxp3 is also expressed in thymic epithelial cells where it is involved in regulation of early thymocyte differentiation and is required to prevent autoimmunity. Here, we used genetic tools to demonstrate that the thymic epithelium does not express Foxp3. Furthermore, we formally showed that genetic abatement of Foxp3 in the hematopoietic compartment, i.e. in T cells, is both necessary and sufficient to induce the autoimmune lesions associated with Foxp3 loss. In contrast, deletion of a conditional Foxp3 allele in thymic epithelial cells did not result in detectable changes in thymocyte differentiation or pathology. Therefore, in mice the only known role for Foxp3 remains promotion of T reg cell differentiation within the T cell lineage, whereas there is no role for Foxp3 in thymic epithelial cells.

The B cell receptor (BCR) is required for stimulation of B cells by antigen, and is also involved in the negative selection of autoreactive B cells. In the past few years, a constitutive ligand-independent signaling activity of the BCR has been demonstrated. In this paper, the various findings are summarized and their interpretation and their significance, both in pathology and in physiology discussed. The constitutive activity of the BCR may be important for tumor formation, at least in the case of heavy-chain diseases, neoplastic proliferations developed from B cells. A large body of evidence suggests that this activity could be required for B cell survival and would play a role in B cell development as a process monitoring BCR functionality. A model explaining signaling in the absence of antigen as a function of dimer formation is proposed. The putative constitutive activity of the pre-BCR is also discussed.

The Protein Tyrosine Phosphatase (PTP) family comprises a large and diverse group of enzymes, regulating a range of biological processes through de-phosphorylation of many proteins and lipids. These enzymes share a catalytic mechanism that requires a reduced and reactive cysteine nucleophile, making them potentially sensitive to inactivation and regulation by oxidation. Analysis of ten PTPs identified substantial differences in the sensitivity of these enzymes to oxidation in vitro. More detailed experiments confirmed the following rank order of sensitivity: PTEN and Sac1>PTPL1/FAP-1>myotubularins. When the apparent sensitivity to oxidation of these PTPs in cells treated with hydrogen peroxide was analysed, this correlated well with the observed sensitivities to oxidation in vitro. These data suggested that different PTPs may fall into at least three different classes with respect to mechanisms of cellular redox regulation. 1. PTEN and Sac1 were readily and reversibly oxidised in vitro and in cells treated with hydrogen peroxide 2. PTPL1 appeared to be resistant to oxidation in cells, correlating with its sensitivity to reduction by glutathione in vitro 3. The myotubularin family of lipid phosphatases was almost completely resistant to oxidation in vitro and in cells. Our results show that sensitivity to reversible oxidation is not a necessary characteristic of the PTPs and imply that such sensitivity has evolved as a regulatory mechanism for some of this large family, but not others.

The pathways regulating integrin-mediated adhesion during neutrophil migration are incompletely defined. Using a flow-based model in which human neutrophils rolling on P-selectin were activated to migrate by the chemoattractant peptide fMLP, we investigated the role of phospholipase D (PLD). fMLP-stimulated PLD generation of phosphatidate (PtdOH); while inhibition of PtdOH production with butan-1-ol had no effect on the initial immobilisation of rolling neutrophils (supported by activation of constitutively surface-expressed beta(2)-integrin CD11b/CD18) it impaired longer-term stability of adhesion and reduced the rate of migration (supported by activation of de novo-exocytosed CD11b/CD18). PtdOH regulated these processes by controlling activation of exocytosed CD11b/CD18, and appeared to act by directly stimulating phosphatidylinositol 4-phosphate 5-kinase type I to generate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Cell-permeable PtdIns(4,5)P(2) recovered migration of neutrophils after PLD inhibition; PtdIns(4,5)P(2) appeared to act by promoting talin binding to CD18 and hence activating CD11b/CD18, as migration was inhibited when neutrophils were loaded with peptides previously shown to block the interaction between PtdIns(4,5)P(2) and talin or talin and CD18. Thus, these data indicate that PLD-synthesised PtdOH stimulates the generation of PtdIns(4,5)P(2), which in turn mediates talin binding to, and activation of, CD11b/CD18 required for neutrophil stable adhesion and migration.

The control of cell growth, that is cell size, is largely controlled by mTOR (the mammalian target of rapamycin), a large serine/threonine protein kinase that regulates ribosome biogenesis and protein translation. mTOR activity is regulated both by the availability of growth factors, such as insulin/IGF-1 (insulin-like growth factor 1), and by nutrients, notably the supply of certain key amino acids. The last few years have seen a remarkable increase in our understanding of the canonical, growth factor-regulated pathway for mTOR activation, which is mediated by the class I PI3Ks (phosphoinositide 3-kinases), PKB (protein kinase B), TSC1/2 (the tuberous sclerosis complex) and the small GTPase, Rheb. However, the nutrient-responsive input into mTOR is important in its own right and is also required for maximal activation of mTOR signalling by growth factors. Despite this, the details of the nutrient-responsive signalling pathway(s) controlling mTOR have remained elusive, although recent studies have suggested a role for the class III PI3K hVps34. In this issue of the Biochemical Journal, Findlay et al. demonstrate that the protein kinase MAP4K3 [mitogen-activated protein kinase kinase kinase kinase-3, a Ste20 family protein kinase also known as GLK (germinal centre-like kinase)] is a new component of the nutrient-responsive pathway. MAP4K3 activity is stimulated by administration of amino acids, but not growth factors, and this is insensitive to rapamycin, most likely placing MAP4K3 upstream of mTOR. Indeed, MAP4K3 is required for phosphorylation of known mTOR targets such as S6K1 (S6 kinase 1), and overexpression of MAP4K3 promotes the rapamycin-sensitive phosphorylation of these same targets. Finally, knockdown of MAP4K3 levels causes a decrease in cell size. The results suggest that MAP4K3 is a new component in the nutrient-responsive pathway for mTOR activation and reveal a completely new function for MAP4K3 in promoting cell growth. Given that mTOR activity is frequently deregulated in cancer, there is much interest in new strategies for inhibition of this pathway. In this context, MAP4K3 looks like an attractive drug target since inhibitors of this enzyme should switch off mTOR, thereby inhibiting cell growth and proliferation, and promoting apoptosis.

Mutations in amyloid precursor protein (APP), tau and apolipoprotein E4 (ApoE4) lead to Alzheimer's disease (AD) or related pathologies. Pathogenesis and interactions between these pathways have been studied in mouse models. Here, we highlight the fact that axons are important sites of cellular pathology in each pathway and propose that pathway convergence at the molecular level might occur in axons. Recent developments suggest that axonal transport of APP influences beta-amyloid deposition and that tau regulates axonal transport. ApoE4 influences both axonal tau phosphorylation and amyloid-induced neurite pathology. Thus, a better understanding of axonal events in AD might help connect the pathogenic mechanisms of beta-amyloid, ApoE4 and tau, indicating the most important steps for therapeutic targeting.

The need to build a tool to facilitate the quick creation and editing of models encoded in the Systems Biology Markup language (SBML) has been growing with the number of users and the increased complexity of the language. SBMLeditor tries to answer this need by providing a very simple, low level editor of SBML files. Users can create and remove all the necessary bits and pieces of SBML in a controlled way, that maintains the validity of the final SBML file.

Regulatory T cells (Tregs) are relatively autoreactive yet, paradoxically, have been found to display normal sensitivity to thymic deletion. The relationship between self-avidity, apoptosis, and the selection of Tregs therefore remains unclear. We show that thymic Tregs develop efficiently, even at low self-avidity, and are moderately resistant to apoptosis in comparison to conventional thymocytes. Consistent with this, although conventional self-reactive T cell populations undergo chronic peripheral deletion, self-reactive Tregs are largely spared removal. Similarly, the distribution of Tregs among peripheral CD4(+) cells exhibits a linear inverse relationship with CD45RB expression, indicating relative apoptosis resistance of Tregs in chronic responses to environmental Ags. We also show that appropriate controls for CD45RB levels are important for comparisons of Treg and conventional T cell activity. When thus controlled, and contrary to previous reports, Tregs exhibit normal sensitivity to cell death through TCR-independent stimuli, such as the purinergic receptor, P2X(7). Finally, although absence of CD45 in gene-targeted mice results in profound T cell hyporesponsiveness, there is little or no effect on thymic Treg frequency. In summary, the data support a model in which signal strength plays little part in Treg lineage specification, though moderate resistance of self-reactive Tregs to apoptosis may result in progressive biasing of peripheral Treg TCRs toward autoreactivity in comparison to those of conventional T cells.

Neurodegenerative disorders involve death of cell bodies, axons, dendrites and synapses, but it is surprisingly difficult to determine the spatiotemporal sequence of events and the causal relationships among these events. Neuronal compartments often crucially depend upon one another for survival, and molecular defects in one compartment can trigger cellular degeneration in distant parts of the neuron. Here, we consider the novel approaches used to understand these biologically complex and technically challenging questions in amyotrophic lateral sclerosis, spinal muscular atrophy, glaucoma, Alzheimer's disease, Parkinson's disease and polyglutamine disorders. We conclude that there is partial understanding of what degenerates first and why, but that controversy remains the rule not the exception. Finally, we highlight strategies for resolving these fundamental issues.