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Trophoblast giant cells are instrumental in promoting blood flow towards the mouse embryo by invading the uterine endometrium and remodelling the maternal vasculature. This process involves the degradation of the perivascular smooth muscle layer and the displacement of vascular endothelial cells to form trophoblast-lined blood sinuses. How this vascular remodelling is achieved at the molecular level remains largely elusive. Here, we show that two placenta-specific cathepsins, Cts7 and Cts8, are expressed in distinct but largely overlapping subsets of giant cells that are in direct contact with maternal arteries. We find that Cts8, but not Cts7, has the capacity to mediate loss of smooth muscle alpha-actin and to disintegrate blood vessels. Consequently, conditional ubiquitous overexpression of Cts8 leads to midgestational embryonic lethality caused by severe vascularization defects. In addition, both cathepsins determine trophoblast cell fate by inhibiting the self-renewing capacity of trophoblast stem cells when overexpressed in vitro. Similarly, transgenic overexpression of Cts7 and Cts8 affects trophoblast proliferation and differentiation by prolonging mitotic cell cycle progression and promoting giant cell differentiation, respectively. We also show that the cell cycle effect is directly caused by some proportion of CTS7 localizing to the nucleus, highlighting the emerging functional diversity of these typically lysosomal proteases in distinct intracellular compartments. Our findings provide evidence for the highly specialized functions of closely related cysteine cathepsin proteases in extra-embryonic development, and reinforce their importance for a successful outcome of pregnancy.

Most neurological diseases are multifactorial diseases, where environmental conditions combine with genetic background or somatic mutations to trigger a pathological state. In the case of Parkinson's Disease and Schizophrenia, recent research revealed that susceptibility genes coded for proteins involved at different steps of specific metabolic networks and cellular processes. Comprehension of the pathology of those diseases is therefore very likely to benefit from Systems approaches. This is also true of their symptomatology, affecting neurological systems at molecular, cellular, and microcircuit levels.

Phagocytosis and activation of the NADPH oxidase are important mechanisms by which neutrophils and macrophages engulf and kill microbial pathogens. We investigated the role of PI3K signaling pathways in the regulation of the oxidase during phagocytosis of Staphylococcus aureus and Escherichia coli by mouse and human neutrophils, a mouse macrophage-like cell line and a human myeloid-like cell line. Phagocytosis of these bacteria was promoted by serum, independent of serum-derived antibodies, and effectively abolished in mouse neutrophils lacking the beta(2)-integrin common chain, CD18. A combination of PI3K isoform-selective inhibitors, mouse knock-outs, and RNA-interference indicated CD18-dependent activation of the oxidase was independent of class I and II PI3Ks, but substantially dependent on the single class III isoform (Vps34). Class III PI3K was responsible for the synthesis of PtdIns(3)P on phagosomes containing either bacteria. The use of mouse neutrophils carrying an appropriate knock-in mutation indicated that PtdIns(3)P binding to the PX domain of their p40(phox) oxidase subunit is important for oxidase activation in response to both S aureus and E coli. This interaction does not, however, account for all the PI3K sensitivity of these responses, particularly the oxidase response to E coli, suggesting that additional mechanisms for PtdIns(3)P-regulation of the oxidase must exist.

PIP4Ks (type II phosphatidylinositol 4-phosphate kinases) are phosphatidylinositol 5-phosphate (PtdIns5P) 4-kinases, believed primarily to regulate cellular PtdIns5P levels. In this study, we investigated the expression, localization, and associated biological activity of the least-studied PIP4K isoform, PIP4Kgamma. Quantitative RT-PCR and in situ hybridization revealed that compared with PIP4Kalpha and PIP4Kbeta, PIP4Kgamma is expressed at exceptionally high levels in the kidney, especially the cortex and outer medulla. A specific antibody was raised to PIP4Kgamma, and immunohistochemistry with this and with antibodies to specific kidney cell markers showed a restricted expression, primarily distributed in epithelial cells in the thick ascending limb and in the intercalated cells of the collecting duct. In these cells, PIP4Kgamma had a vesicular appearance, and transfection of kidney cell lines revealed a partial Golgi localization (primarily the matrix of the cis-Golgi) with an additional presence in an unidentified vesicular compartment. In contrast to PIP4Kalpha, bacterially expressed recombinant PIP4Kgamma was completely inactive but did have the ability to associate with active PIP4Kalpha in vitro. Overall our data suggest that PIP4Kgamma may have a function in the regulation of vesicular transport in specialized kidney epithelial cells.

Regulatory T (T reg) cells are indispensable for preventing autoimmunity. Incumbent to this role is the ability of T reg cells to exert their suppressor function under inflammatory conditions. We found that T reg cell-mediated tolerance is critically dependent on the Dicer-controlled microRNA (miRNA) pathway. Depletion of miRNA within the T reg cell lineage resulted in fatal autoimmunity indistinguishable from that in T reg cell-deficient mice. In disease-free mice lacking Dicer in all T cells or harboring both Dicer-deficient and -sufficient T reg cells, Dicer-deficient T reg cells were suppressive, albeit to a lesser degree, whereas their homeostatic potential was diminished as compared with their Dicer-sufficient counterparts. However, in diseased mice, Dicer-deficient T reg cells completely lost suppressor capacity. Thus, miRNA preserve the T reg cell functional program under inflammatory conditions.

ATP-gated P2X receptors (P2XRs) are ligand-gated ion channels (LGICs) presumably trimeric. To date, no experimental high-resolution structures are available. Recent X-ray structure of the acid-sensing ion channel 1 (ASIC1) revealed an unexpected trimeric ion channel. Beside their quaternary structure, P2XR and ASIC1 share common membrane topologies, but no significant sequence similarity. In order to overcome this low sequence resemblance, we have developed comparative models of P2X(2)R based on secondary structure predictions using the crystal structure of ASIC1 as template. These models were constrained to be consistent with known arrangement of disulfide bridges. They agreed with cross-linking experiments and supported inter-subunit ATP-binding sites. One of our models reconciled most existing data and provides new structural insights for a plausible mechanism of gating, thus encouraging new experiments.

Regulatory Foxp3(+) T cells (T(R)) are indispensable for preventing autoimmune pathology in multiple organs and tissues. During thymic differentiation T cell receptor (TCR)-ligand interactions within a certain increased affinity range, in conjunction with gammac-containing cytokine receptor signals, induce Foxp3 expression and thereby commit developing thymocytes to the T(R) lineage. The contribution of distinct MHC class II-expressing accessory cell types to the differentiation process of Foxp3(+) thymocytes remains controversial, because a unique role in this process has been ascribed to either thymic dendritic cells (tDC) or to medullary thymic epithelial cells (mTEC). Furthermore, it was suggested that the thymic medulla, where the bulk of the negative selection of self-reactive thymocytes takes place, provides a specialized microenvironment supporting T(R) differentiation. Here, we report that the cortex, as defined by cortical thymic epithelial cells (cTEC), is sufficient for supporting T(R) differentiation. MHC class II expression restricted to both cTEC and mTEC or to cTEC alone did not significantly affect the numbers of Foxp3(+) thymocytes. Furthermore, genetic or pharmacologic blockade of thymocyte migration resulted in a prominent accumulation of Foxp3(+) thymocytes in the cortex, demonstrating that secondary signals required for Foxp3 up-regulation exist in the cortex. Our results suggest that mTEC or tDC do not serve as a cell type singularly responsible for T(R) differentiation and that neither the cortex nor the medulla exclusively provides an environment suitable for Foxp3 induction. Instead, multiple accessory cell types probably contribute to the thymic generation of regulatory Foxp3(+) T cells.

Unlike most other species, ram spermatozoa are difficult to capacitate in vitro. Bicarbonate and Ca(2+) are necessary, whereas bovine serum albumin does not appear to be obligatory. In the present investigation we have assessed (1) the ability of the cholesterol-sequestering agent, methyl-beta-cyclodextrin (M-beta-CD), to initiate protein tyrosine phosphorylation, and (2) the importance of phosphodiesterases (PDEs) in controlling the levels of cAMP. Results show that despite removing significant amounts of membrane cholesterol, as assessed by filipin staining, M-beta-CD treatment did not stimulate major increases in protein tyrosine phosphorylation. Addition of a cocktail of PDE inhibitors (theophylline and caffeine), a phosphatase inhibitor (okadaic acid) and dibutyryl-cAMP (db-cAMP), however, stimulated specific tyrosine phosphorylation of several proteins between 30 and 120 kDa. On their own, none of the above reagents were effective but a combination of db-cAMP + PDE inhibitors was sufficient to achieve a maximal response. H-89, a protein kinase-A inhibitor, suppressed tyrosine phosphorylation significantly. Immunofluorescence revealed that the newly-phosphorylated proteins localised mainly in the sperm tail. These findings suggest that in ram spermatozoa cAMP levels are too low to initiate tyrosine phosphorylation of flagellar proteins that are indicative of the capacitation state and that this is caused by unusually high levels of intracellular PDEs.

Vascular calcification is associated with an increased risk of myocardial infarction; however, the mechanisms linking these 2 processes are unknown. Studies in macrophages have suggested that calcium phosphate crystals induce the release of proinflammatory cytokines; however, no studies have been performed on the effects of calcium phosphate crystals on vascular smooth muscle cell function. In the present study, we found that calcium phosphate crystals induced cell death in human aortic vascular smooth muscle cells with their potency depending on their size and composition. Calcium phosphate crystals of approximately 1 microm or less in diameter caused rapid rises in intracellular calcium concentration, an effect that was inhibited by the lysosomal proton pump inhibitor, bafilomycin A1. Bafilomycin A1 also blocked vascular smooth muscle cell death suggesting that crystal dissolution in lysosomes leads to an increase in intracellular calcium levels and subsequent cell death. These studies give novel insights into the bioactivity of calcified deposits and suggest that small calcium phosphate crystals could destabilize atherosclerotic plaques by initiating inflammation and by causing vascular smooth muscle cell death.

Calmodulin plays a vital role in mediating bidirectional synaptic plasticity by activating either calcium/calmodulin-dependent protein kinase II (CaMKII) or protein phosphatase 2B (PP2B) at different calcium concentrations. We propose an allosteric model for calmodulin activation, in which binding to calcium facilitates the transition between a low-affinity [tense (T)] and a high-affinity [relaxed (R)] state. The four calcium-binding sites are assumed to be nonidentical. The model is consistent with previously reported experimental data for calcium binding to calmodulin. It also accounts for known properties of calmodulin that have been difficult to model so far, including the activity of nonsaturated forms of calmodulin (we predict the existence of open conformations in the absence of calcium), an increase in calcium affinity once calmodulin is bound to a target, and the differential activation of CaMKII and PP2B depending on calcium concentration.

The conditional kinase DeltaMEKK3:ER allows activation of JNK, p38 and ERK1/2 without overt cellular stress or damage and has proved useful in understanding how these pathways regulate apoptosis and cell cycle progression. We have previously shown that activation of DeltaMEKK3:ER causes a sustained G(1) cell cycle arrest which requires p21(CIP1), with ERK1/2 and p38 cooperating to promote p21(CIP1) expression. In cells lacking p21(CIP1), DeltaMEKK3:ER causes only a transient delay in cell cycle re-entry. We now show that this delay in cell cycle re-entry is due to a reduction in cyclin D1 levels. Activation of DeltaMEKK3:ER promotes the proteasome-dependent turnover of cyclin D1; this requires phosphorylation of threonine 286 (T(286)) and expression of cyclin D1T(286)A rescues the delay in G(1)/S progression. DeltaMEKK3:ER-dependent phosphorylation of T(286) does not appear to be mediated by GSK3beta but requires activation of the ERK1/2 and p38 pathways. ERK1/2 can physically associate with cyclin D1 but activation of ERK1/2 alone is not sufficient for phosphorylation of T(286). Rather, cyclin D1 phosphorylation appears to require coincident activation of ERK1/2 and p38. Thus activation of DeltaMEKK3:ER promotes a sustained G(1) cell cycle arrest by a bipartite mechanism involving the rapid destruction of cyclin D1 and the slower more prolonged expression of p21(CIP1). This has parallels with the bipartite response to ionizing radiation and p53-independent mechanisms of G(1) cell cycle arrest in simple organisms such as yeast.

The antiapoptotic protein Bcl-2 inhibits Ca2+ release from the endoplasmic reticulum (ER). One proposed mechanism involves an interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel localized with Bcl-2 on the ER. Here we document Bcl-2-IP3R interaction within cells by FRET and identify a Bcl-2 interacting region in the regulatory and coupling domain of the IP3R. A peptide based on this IP3R sequence displaced Bcl-2 from the IP3R and reversed Bcl-2-mediated inhibition of IP3R channel activity in vitro, IP3-induced ER Ca2+ release in permeabilized cells, and cell-permeable IP3 ester-induced Ca2+ elevation in intact cells. This peptide also reversed Bcl-2's inhibition of T cell receptor-induced Ca2+ elevation and apoptosis. Thus, the interaction of Bcl-2 with IP3Rs contributes to the regulation of proapoptotic Ca2+ signals by Bcl-2, suggesting the Bcl-2-IP3R interaction as a potential therapeutic target in diseases associated with Bcl-2's inhibition of cell death.

The Cdx2 homeobox gene exerts multiple functions including trophectoderm specification, antero-posterior patterning, and determination of intestinal identity. The aim of this study was to map genomic regions that regulate the transcription of Cdx2, with a particular interest in the gut.

Experimental reduction in placental growth often leads to increased placental efficiency measured as grams of fetus produced per gram of placenta, although little is known about the mechanisms involved. This study tested the hypothesis that the smallest placenta within a litter is the most efficient at supporting fetal growth by examining the natural intra-litter variation in placental nutrient transfer capacity in normal pregnant mice. The morphology, nutrient transfer and expression of key growth and nutrient supply genes (Igf2P0, Grb10, Slc2a1, Slc2a3, Slc38a1, Slc38a2 and Slc38a4) were compared in the lightest and heaviest placentas of a litter at days 16 and 19 of pregnancy, when mouse fetuses are growing most rapidly in absolute terms. The data show that there are morphological and functional adaptations in the lightest placenta within a litter, which increase active transport of amino acids per gram of placenta and maintain normal fetal growth close to term, despite the reduced placental mass. The specific placental adaptations differ with age. At E16, they are primarily morphological with an increase in the volume fraction of the labyrinthine zone responsible for nutrient exchange, whereas at E19 they are more functional with up-regulated placental expression of the glucose transporter gene, Slc2a1/GLUT1 and one isoform the System A family of amino acid transporters, Slc38a2/SNAT2. Thus, this adaptability in placental phenotype provides a functional reserve capacity for maximizing fetal growth during late gestation when placental growth is compromised.

The vesicular monoamine transporter 2 (VMAT2) controls the loading of dopamine (DA) into vesicles and therefore determines synaptic properties such as quantal size, receptor sensitivity, and vesicular and cytosolic DA concentration. Impairment of proper DA compartmentalization is postulated to underlie the sensitivity of DA neurons to oxidative damage and degeneration. It is known that DA can auto-oxidize in the cytosol to form quinones and other oxidative species and that this production of oxidative stress is thought to be a critical factor in DA terminal loss after methamphetamine (METH) exposure. Using a mutant strain of mice (VMAT2 LO), which have only 5-10% of the VMAT2 expressed by wild-type animals, we show that VMAT2 is a major determinant of METH toxicity in the striatum. Subsequent to METH exposure, the VMAT2 LO mice show an exacerbated loss of dopamine transporter and tyrosine hydroxylase (TH), as well as enhanced astrogliosis and protein carbonyl formation. More importantly, VMAT2 LO mice show massive argyrophilic deposits in the striatum after METH, indicating that VMAT2 is a regulator of METH-induced neurodegeneration. The increased METH neurotoxicity in VMAT2 LO occurs in the absence of any significant difference in basal temperature or METH-induced hyperthermia. Furthermore, primary midbrain cultures from VMAT2 LO mice show more oxidative stress generation and a greater loss of TH positive processes than wild-type cultures after METH exposure. Elevated markers of neurotoxicity in VMAT2 LO mice and cultures suggest that the capacity to store DA determines the amount of oxidative stress and neurodegeneration after METH administration.

When young suckle, they are rewarded intermittently with a let-down of milk that results from reflex secretion of the hormone oxytocin; without oxytocin, newly born young will die unless they are fostered. Oxytocin is made by magnocellular hypothalamic neurons, and is secreted from their nerve endings in the pituitary in response to action potentials (spikes) that are generated in the cell bodies and which are propagated down their axons to the nerve endings. Normally, oxytocin cells discharge asynchronously at 1-3 spikes/s, but during suckling, every 5 min or so, each discharges a brief, intense burst of spikes that release a pulse of oxytocin into the circulation. This reflex was the first, and is perhaps the best, example of a physiological role for peptide-mediated communication within the brain: it is coordinated by the release of oxytocin from the dendrites of oxytocin cells; it can be facilitated by injection of tiny amounts of oxytocin into the hypothalamus, and it can be blocked by injection of tiny amounts of oxytocin antagonist. Here we show how synchronized bursting can arise in a neuronal network model that incorporates basic observations of the physiology of oxytocin cells. In our model, bursting is an emergent behaviour of a complex system, involving both positive and negative feedbacks, between many sparsely connected cells. The oxytocin cells are regulated by independent afferent inputs, but they interact by local release of oxytocin and endocannabinoids. Oxytocin released from the dendrites of these cells has a positive-feedback effect, while endocannabinoids have an inhibitory effect by suppressing the afferent input to the cells.

Cellular identity is determined by the switching on and off of lineage-specific genes. This dynamic process is regulated by a highly co-ordinated series of chromatin remodelling mechanisms that control DNA accessibility to facilitate transcription, replication and recombination. The identity of an individual B-lymphocyte is defined by the expression of a unique antibody protein, composed of two identical immunoglobulin heavy and two identical light chain polypeptides, which recognize a single foreign antigen with high specificity. However, the mammalian adaptive immune system requires an enormous variety of antibody-expressing B cells to combat the millions of foreign antigens it may encounter. This diversity is generated primarily at the multigene immunoglobulin loci by V(D)J recombination, a specialised form of DNA recombination in which numerous variable (V), diversity (D) and joining (J) genes are cut and pasted together in a strict order to allow shuffling of immunoglobulin genes. The mouse immunoglobulin heavy chain (Igh) locus is the largest known multigene locus. It spans approximately 3 Mb and comprises more than 200 genes. Its size and complexity pose an enormous logistic challenge to the chromatin remodelling machinery, but recent major advances in our understanding of how the 200 genes are shuffled have begun to reveal an exquisitely co-ordinated set of chromatin remodelling mechanisms which exploit every aspect of nuclear dynamics, and provide a global view of multigene regulation. This review will explore the numerous processes implicated in opening up and positioning of the locus to enable shuffling of the Igh locus genes, including non-coding RNA transcription, histone modifications, transcription factors, nuclear relocation and locus contraction.

There is increasing evidence that different transcription units are transcribed together in discrete nuclear structures known as transcription factories. Various new techniques enable us to detect and characterize these structures. We review the latest findings and discuss how they support a model for transcription and chromosome organization.

Long-distance chromosomal interactions are emerging as a potential mechanism of gene expression control. In this issue, Apostolou and Thanos (2008) describe how viral infection elicits interchromosomal associations between the interferon-beta (IFN-beta) gene enhancer and DNA binding sites of the transcription factor NF-kappaB, resulting in the initiation of transcription and an antiviral response.

Inositol 1,4,5-trisphosphate (IP(3)) is a ubiquitous intracellular messenger regulating diverse functions in almost all mammalian cell types. It is generated by membrane receptors that couple to phospholipase C (PLC), an enzyme which liberates IP(3) from phosphatidylinositol 4,5-bisphosphate (PIP(2)). The major action of IP(3), which is hydrophilic and thus translocates from the membrane into the cytoplasm, is to induce Ca(2+) release from endogenous stores through IP(3) receptors (IP(3)Rs). Cardiac excitation-contraction coupling relies largely on ryanodine receptor (RyR)-induced Ca(2+) release from the sarcoplasmic reticulum. Myocytes express a significantly larger number of RyRs compared to IP(3)Rs (~100:1), and furthermore they experience substantial fluxes of Ca(2+) with each heartbeat. Therefore, the role of IP(3) and IP(3)-mediated Ca(2+) signaling in cardiac myocytes has long been enigmatic. Recent evidence, however, indicates that despite their paucity cardiac IP(3)Rs may play crucial roles in regulating diverse cardiac functions. Strategic localization of IP(3)Rs in cytoplasmic compartments and the nucleus enables them to participate in subsarcolemmal, bulk cytoplasmic and nuclear Ca(2+) signaling in embryonic stem cell-derived and neonatal cardiomyocytes, and in adult cardiac myocytes from the atria and ventricles. Intriguingly, expression of both IP(3)Rs and membrane receptors that couple to PLC/IP(3) signaling is altered in cardiac disease such as atrial fibrillation or heart failure, suggesting the involvement of IP(3) signaling in the pathology of these diseases. Thus, IP(3) exerts important physiological and pathological functions in the heart, ranging from the regulation of pacemaking, excitation-contraction and excitation-transcription coupling to the initiation and/or progression of arrhythmias, hypertrophy and heart failure.

Comparisons between eutherians and marsupials suggest limited conservation of the molecular mechanisms that control genomic imprinting in mammals. We have studied the evolution of the imprinted IGF2-H19 locus in therians. Although marsupial orthologs of protein-coding exons were easily identified, the use of evolutionarily conserved regions and low-stringency Bl2seq comparisons was required to delineate a candidate H19 noncoding RNA sequence. The therian H19 orthologs show miR-675 and exon structure conservation, suggesting functional selection on both features. Transcription start site sequences and poly(A) signals are also conserved. As in eutherians, marsupial H19 is maternally expressed and paternal methylation upstream of the gene originates in the male germline, encompasses a CTCF insulator, and spreads somatically into the H19 gene. The conservation in all therians of the mechanism controlling imprinting of the IGF2-H19 locus suggests a sequential model of imprinting evolution.

Nascent, full length, immunoglobulin (Ig) heavy (H)-chains are post-translationally associated with H-chain-binding protein (BiP or GRP78) in the endoplasmic reticulum (ER). The first constant (C) domain, CH1 of a C gene (Cmu, Cgamma, Calpha), is important for this interaction. The contact is released upon BiP replacement by conventional Ig light (L)-chain (kappa or lambda). Incomplete or mutated H-chains with removed variable (VH) and/or C(H)1 domain, as found in H-chain disease (HCD), can preclude stable BiP interaction. Progression in development after the preB cell stage is dependent on surface expression of IgM when association of a micro H-chain with a L-chain overcomes the retention by BiP. We show that IgM lacking the BiP-binding domain is displayed on the cell surface and elicits a signal that allows developmental progression even without the presence of L-chain. The results are reminiscent of single chain Ig secretion in camelids where developmental processes leading to the generation of fully functional H-chain-only antibodies are not understood. Furthermore, in the mouse the largest secondary lymphoid organ, the spleen, is not required for H-chain-only Ig expression and the CD5 survival signal may be obsolete for cells expressing truncated IgM.