Dynamic interactions of cells with their environment regulate multiple aspects of tissue morphogenesis and function. Integrins are the major class of cell surface receptors that recognize and bind extracellular matrix proteins, resulting in the engagement and organization of the cytoskeleton as well as activation of signalling pathways to regulate cell behaviour and morphogenetic processes. The ternary complex of integrin-linked kinase (ILK), PINCH, and parvin (IPP complex), which was identified more than a decade ago, interacts with the cytoplasmic tail of beta integrins and couples them to the actin cytoskeleton. In addition, ILK has been shown to act as a serine/threonine kinase and to directly activate several signalling pathways downstream of integrins. However, the kinase activity of ILK and the precise functions of the IPP complex have remained elusive and controversial. This review focuses on the recent advances made towards understanding the specialized roles this complex and its individual components have acquired during evolution.

Meningiomas, one of the most common human brain tumors, are derived from arachnoidal cells associated with brain meninges, are usually benign, and are frequently associated with neurofibromatosis type 2. Here, we define a typical human meningioma microRNA (miRNA) profile and characterize the effects of one downregulated miRNA, miR-200a, on tumor growth. Elevated levels of miR-200a inhibited meningioma cell growth in culture and in a tumor model in vivo. Upregulation of miR-200a decreased the expression of transcription factors ZEB1 and SIP1, with consequent increased expression of E-cadherin, an adhesion protein associated with cell differentiation. Downregulation of miR-200a in meningiomas and arachnoidal cells resulted in increased expression of beta-catenin and cyclin D1 involved in cell proliferation. miR-200a was found to directly target beta-catenin mRNA, thereby inhibiting its translation and blocking Wnt/beta-catenin signaling, which is frequently involved in cancer. A direct correlation was found between the downregulation of miR-200a and the upregulation of beta-catenin in human meningioma samples. Thus, miR-200a appears to act as a multifunctional tumor suppressor miRNA in meningiomas through effects on the E-cadherin and Wnt/beta-catenin signaling pathways. This reveals a previously unrecognized signaling cascade involved in meningioma tumor development and highlights a novel molecular interaction between miR-200a and Wnt signaling, thereby providing insights into novel therapies for meningiomas.

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide. Discovered 30 years ago, it is produced as a consequence of alternative RNA processing of the calcitonin gene. CGRP has two major forms (α and β). It belongs to a group of peptides that all act on an unusual receptor family. These receptors consist of calcitonin receptor-like receptor (CLR) linked to an essential receptor activity modifying protein (RAMP) that is necessary for full functionality. CGRP is a highly potent vasodilator and, partly as a consequence, possesses protective mechanisms that are important for physiological and pathological conditions involving the cardiovascular system and wound healing. CGRP is primarily released from sensory nerves and thus is implicated in pain pathways. The proven ability of CGRP antagonists to alleviate migraine has been of most interest in terms of drug development, and knowledge to date concerning this potential therapeutic area is discussed. Other areas covered, where there is less information known on CGRP, include arthritis, skin conditions, diabetes, and obesity. It is concluded that CGRP is an important peptide in mammalian biology, but it is too early at present to know if new medicines for disease treatment will emerge from our knowledge concerning this molecule.

The Arabidopsis gene SUPERMAN (SUP) is necessary for the proper spatial development of reproductive floral tissues. Recessive mutations cause extra stamens to form interior to the normal third whorl stamens, at the expense of fourth whorl carpel development. The mutant phenotype is associated with the ectopic expression of the B function genes, AP3 and PI, in the altered floral region, closer to the centre of the flower than in the wild type, and ap3 sup and pi sup double mutants exhibit a phenotype similar to ap3 and pi single mutants. These findings led to SUP being interpreted as an upstream negative regulator of the B function organ-identity genes, acting in the fourth whorl, to establish a boundary between stamen and carpel whorls. Here we show, using molecular cloning and analysis, that it is expressed in the third whorl and acts to maintain this boundary in developing flowers. The putative SUPERMAN protein contains one zinc-finger and a region resembling a basic leucine zipper motif, suggesting a function in transcriptional regulation.

Fragments of the cloned hepatitis B virus (HBV) genome were assayed in vivo for the presence of a transcriptional enhancer element. We demonstrate that sequences positioned approximately 450 bp upstream from the HBcAg gene promoter are required for its efficient activity. These HBV stimulatory sequences activate transcription when inserted upstream to a heterologous SV40 early promoter. Like other known enhancer elements, this HBV sequence acts in an orientation-independent manner. Furthermore, the HBV enhancer element exhibits a preferred activity in a human hepatoma cell line.

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.

Tight junctions in epithelial cells have been postulated to act as barriers inhibiting lateral diffusion of lipids and proteins between the apical and basolateral plasma membrane domains. To study the fence function of the tight junction in more detail, we have fused liposomes containing the fluorescent phospholipid N-Rh-PE into the apical plasma membrane of MDCK cells. Liposome fusion was induced by low pH and mediated by the influenza virus hemagglutinin, which was expressed on the apical cell surface after viral infection. Redistribution of N-Rh-PE to the basolateral surface, monitored at 0 degree C by fluorescence microscopy, appeared to be dependent on the transbilayer orientation of the fluorescent lipids in the plasma membrane. Asymmetric liposomes containing over 85% of the N-Rh-PE in the external bilayer leaflet, as shown by a phospholipase A2 assay, were generated by octyl beta-D-glucoside dialysis. When these asymmetric liposomes were fused with the apical plasma membrane, fluorescent lipid did not move to the basolateral side. Symmetric liposomes which contained the marker in both leaflets were obtained by freeze-thawing asymmetric liposomes or by reverse-phase evaporation. Upon fusion of these with the apical membrane, redistribution to the basolateral membrane occurred immediately. Redistribution could be observed with asymmetric liposomes only when the tight junctions were opened by incubation in a Ca2+-free medium. During the normal experimental manipulations the tight junctions remained intact since a high trans-epithelial electrical resistance was maintained over the cell monolayer. We conclude that the tight junction acts as a diffusion barrier for the fluorescent phospholipid N-Rh-PE in the exoplasmic leaflet of the plasma membrane but not in the cytoplasmic leaflet.

Morbilliviruses comprise measles virus, canine distemper virus, rinderpest virus, and several other viruses that cause devastating human and animal diseases accompanied by severe immunosuppression and lymphopenia. Recently, we have shown that human signaling lymphocyte activation molecule (SLAM) is a cellular receptor for measles virus. In this study, we examined whether canine distemper and rinderpest viruses also use canine and bovine SLAMs, respectively, as cellular receptors. The Onderstepoort vaccine strain and two BB cell line)-isolated strains of canine distemper virus caused extensive cytopathic effects in normally resistant CHO (Chinese hamster ovary) cells after expression of canine SLAM. The Ako vaccine strain of rinderpest virus produced strong cytopathic effects in bovine SLAM-expressing CHO cells. The data on entry with vesicular stomatitis virus pseudotypes bearing measles, canine distemper, or rinderpest virus envelope proteins were consistent with development of cytopathic effects in SLAM-expressing CHO cell clones after infection with the respective viruses, confirming that SLAM acts at the virus entry step (as a cellular receptor). Furthermore, most measles, canine distemper, and rinderpest virus strains examined could any use of the human, canine, and bovine SLAMs to infect cells. Our findings suggest that the use of SLAM as a cellular receptor may be a property common to most, if not all, morbilliviruses and explain the lymphotropism and immunosuppressive nature of morbilliviruses.

By virtue of its control over major histocompatibility complex class II (MHC-II) gene expression, CIITA represents a key molecule in the regulation of adaptive immune responses. It was first identified as a factor that is defective in MHC-II deficiency, a hereditary disease characterized by the absence of MHC-II expression. CIITA is a highly regulated transactivator that governs all spatial, temporal, and quantitative aspects of MHC-II expression. It has been proposed to act as a non-DNA-binding transcriptional coactivator, but evidence that it actually functions at the level of MHC-II promoters was lacking. By means of chromatin immunoprecipitation assays, we show here for the first time that CIITA is physically associated with MHC-II, as well as HLA-DM, Ii, MHC-I, and beta(2)m promoters in vivo. To dissect the mechanism by which CIITA is recruited to the promoter, we have developed a DNA-dependent coimmunoprecipitation assay and a pull-down assay using immobilized promoter templates. We demonstrate that CIITA recruitment depends on multiple, synergistic protein-protein interactions with DNA-bound factors constituting the MHC-II enhanceosome. CIITA therefore represents a paradigm for a novel type of regulatory and gene-specific transcriptional cofactor.

Systemic lupus erythematosus, Sjögren's syndrome, and dermatomyositis are systemic autoimmune diseases that develop after environmental triggering of genetically susceptible individuals. The precise cellular and molecular mechanisms leading to autoimmune disease, and what factors determine which organs are involved, remain poorly understood. Recent insights into genetic susceptibility now make obvious that environmental triggers often act via cellular pathways containing disease-associated polymorphisms. In the breaking of tolerance, the initiating tissue--including dendritic cells--provides a decisive microenvironment that affects immune-cell differentiation, leading to activation of adaptive immunity. Type 1 interferon produced by innate immune cells has a central role in systemic autoimmunity and activates B cells and T cells. In turn, B-cell-derived autoantibodies stimulate dendritic cells to produce type 1 interferon; thus, a positive feedforward loop is formed that includes both the innate and adaptive systems. New treatments could simultaneously and specifically target several such vital pathways in autoimmunity.

Previous studies have proposed that oxygen radicals may play a role in the triggering of ischemic preconditioning. However, studies evaluating the effects of radical scavengers have yielded conflicting results, possibly because of differences in the number of preconditioning episodes used. The present study tested whether N-2-mercaptopropionylglycine (MPG) could block protection of both single and multiple episodes of preconditioning in in situ and in vitro rabbit hearts. All hearts were subjected to 30 min of regional ischemia followed by reperfusion for 2 (in vitro) or 3 (in situ) h. Infarct size was measured by tetrazolium. Infarction in control in situ hearts was 37.5+/-3.5% of the risk zone. A single cycle of preconditioning (PC1), with 5 min ischemia/10 min reperfusion, reduced infarct size to 12.3+/-2.0% (P<0.05). Four cycles of preconditioning (PC4) were equally protective. MPG (1 mg/kg/min i.v.) alone had no effect on infarction but abolished protection afforded by PC1 (35.4+/-3.9%). However, MPG failed to block protection in the PC4 group. In isolated control hearts, infarct size was 31.1+/-1.8% and was reduced to 10.2+/-2.2% (P<0.05) by preconditioning. MPG (300 microM) aborted protection. Infusion of hypoxanthine or xanthine oxidase separately in lieu of preconditioning had no effect on infarct size, but induced protection when combined (14.1+/-2.2%; P<0.05). Polymyxin B, an inhibitor of protein kinase C (PKC), abolished this protection (53.1+/-4.1%). In conclusion, oxygen radicals contribute to ischemic preconditioning in the rabbit and appear to do so via activation of PKC. The fact that MPG could not block protection by PC4 suggests that oxygen radicals act in concert with other triggers of preconditioning such as adenosine and bradykinin.

Microglial cells are believed to play an active role in brain inflammatory, immune and degenerative processes. Depending on the magnitude of microglial reaction, on the type of stimulus and on the concurrence of other local factors, microglia can contribute to host defence and repair, or to the establishment and maintenance of brain damage. Many of the effects of microglial cells can be ascribed to the numerous substances that these cells can synthesize and release in response to a variety of stimuli (cytokines, pro-inflammatory substances, neurotransmitters, toxins, etc.). The present article deals with two classes of compounds that activated microglial cells can produce in large amounts: prostanoids (that derive from arachidonic acid through the cyclooxygenase pathway), and nitric oxide (that is synthesized from arginine by nitric oxide synthase). Prostanoids and nitric oxide have a number of common targets, on which they may exert similar or opposite actions, and have a crucial role in the regulation of inflammation, immune responses and cell viability. Their synthesis can massively increase when the inducible isoforms of cyclooxygenase and nitric oxide synthase are expressed. The metabolic pathways of prostanoids and nitric oxide are finely tuned by the respective end-products, by cyclic AMP and by a number of exogenous factors, such as cytokines, glucocorticoids, lipocortin-1 and others. Some of these factors (e.g. transforming growth factor-beta 1, interleukin-10, lipocortin-1) may be secreted by microglial cells themselves, and act in an autocrine-paracrine way. In view of the neuroprotective role attributed to some prostaglandins and to the cytotoxicity of excessive levels of nitric oxide or its derivatives, the balance between prostanoid and nitric oxide levels may be crucial for orienting microglial reactions towards neuroprotection or neurotoxicity.

The WalK/WalR (aka YycG/YycF) two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and specific to low G+C Gram-positive bacteria, including a number of important pathogens. An unusual feature is that this system is essential for viability in most of these bacteria. Recent studies have revealed conserved functions for this system, defining this signal transduction pathway as a crucial regulatory system for cell wall metabolism, that we have accordingly renamed WalK/WalR. Here we review the cellular role of the WalK/WalR TCS in different bacterial species, focusing on the function of genes in its regulon, as well as variations in walRK operon structure and the composition of its regulon. We also discuss the nature of its essentiality and the potential type of signal being sensed. The WalK histidine kinase of B. subtilis has been shown to localize to the divisome and we suggest that the WalKR system acts as an information conduit between extracytoplasmic cellular structures and intracellular processes required for their synthesis, playing a vital role in effectively co-ordinating peptidoglycan plasticity with the cell division process.

OBJECTIVE

Ivabradine, a new I(f) inhibitor which acts specifically on the pacemaker activity of the sinoatrial node, is a pure heart rate lowering agent. Ivabradine has shown anti-ischaemic and anti-anginal activity in a placebo-controlled trial. The objective of this study was to compare the anti-anginal and anti-ischaemic effects of ivabradine and the beta-blocker atenolol.

RESULTS

In a double-blinded trial, 939 patients with stable angina were randomized to receive ivabradine 5 mg bid for 4 weeks and then either 7.5 or 10 mg bid for 12 weeks or atenolol 50 mg od for 4 weeks and then 100 mg od for 12 weeks. Patients underwent treadmill exercise tests at randomization (M(0)) and after 4 (M(1)) and 16 (M(4)) weeks of therapy. Increases in total exercise duration (TED) at trough at M(4) were 86.8+/-129.0 and 91.7+/-118.8 s with ivabradine 7.5 and 10 mg, respectively and 78.8+/-133.4 s with atenolol 100 mg. Mean differences (SE) when compared with atenolol 100 mg were 10.3 (9.4) and 15.7 (9.5) s in favour of ivabradine 7.5 and 10 mg (P<0.001 for non-inferiority). TED at M(1) improved by 64.2+/-104.0 s with ivabradine 5 mg and by 60.0+/-114.4 s with atenolol 50 mg (P<0.001 for non-inferiority). Non-inferiority of ivabradine was shown at all doses and for all criteria. The number of angina attacks was decreased by two-thirds with both ivabradine and atenolol.

CONCLUSIONS

Ivabradine is as effective as atenolol in patients with stable angina.

The anterior heart field (AHF), which contributes to the outflow tract and right ventricle of the heart, is defined in part by expression of the LIM homeobox transcription factor Isl-1. The importance of Isl-1-positive cells in cardiac development and homeostasis is underscored by the finding that these cells are required for cardiac development and act as cardiac stem/progenitor cells within the postnatal heart. However, the molecular pathways regulating these cells' expansion and differentiation are poorly understood. We show that Isl-1-positive AHF progenitor cells in mice were responsive to Wnt/beta-catenin signaling, and these responsive cells contributed to the outflow tract and right ventricle of the heart. Loss of Wnt/beta-catenin signaling in the AHF caused defective outflow tract and right ventricular development with a decrease in Isl-1-positive progenitors and loss of FGF signaling. Conversely, Wnt gain of function in these cells led to expansion of Isl-1-positive progenitors with a concomitant increase in FGF signaling through activation of a specific set of FGF ligands including FGF3, FGF10, FGF16, and FGF20. These data reveal what we believe to be a novel Wnt-FGF signaling axis required for expansion of Isl-1-positive AHF progenitors and suggest future therapies to increase the number and function of these cells for cardiac regeneration.

Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds.

In skin, the profibrotic protein connective tissue growth factor (CTGF) is not normally expressed. However, when skin cells are exposed to transforming growth factor-beta (TGF-beta), CTGF is induced in fibroblasts but not in epithelial cells. We have begun to investigate the requirements for the fibroblast-selective induction of CTGF by TGF-beta. Previously we found that this response was Smad-dependent. Now we show that protein kinase C and Ras/MEK/ERK are necessary for the TGF-beta induction of the CTGF promoter but not of a generic Smad-responsive promoter (SBE-lux). Induction of the CTGF promoter is antagonized by c-Jun or by MEKK1, suggesting that a proper balance between the Ras/MEK/ERK and JNK MAPK cascades is necessary for TGF-beta induction of CTGF. We identify the minimal CTGF promoter element necessary and sufficient to confer TGF-beta responsiveness to a heterologous promoter and show that a tandem repeat of a consensus transcription enhancer factor binding element, 5'-GAGGAATGG-3', is necessary for this induction. This element has not been previously shown to play a role in TGF-beta induction of gene expression in fibroblasts. Gel shift analysis shows that this sequence binds nuclear factors that are greatly enriched in fibroblasts relative to epithelial cells. Thus Smads, Ras/MEK/ERK, protein kinase C, and fibroblast-enriched factors that bind GAGGAATGG act together to drive the TGF-beta-mediated induction of CTGF in fibroblasts.

Asthma is a complex clinical syndrome with multiple genetic and environmental factors contributing to its phenotypic expression. This aetiological heterogeneity adds to the complexity when addressing variation in the response to anti-asthma treatment. Currently, there are three main lines of treatment available: (i) inhaled glucocorticoids which have multiple mechanisms of action; (ii) beta 2-agonists which are very effective bronchodilators and act predominantly on airway smooth muscle; and (iii) cysteinyl-leukotriene inhibitors. Analysis of the repeatability (r) of the treatment response, defined as the fraction of the total population variance which results from among-individual differences, shows values of r between 60-80% indicating that a substantial fraction of the variance of the treatment response could be genetic in nature. Among the sources of variability that could contribute to the observed heterogeneity in the response to treatment are the degree of underlying inflammation, such as in glucocorticoid resistance, and polymorphisms in the genes encoding the drug target, such as beta 2-adrenoceptor and 5-lipoxygenase.

Cathelicidins (caths) are peptides that are expressed at high levels in neutrophils and some epithelia and can act as natural antibiotics by directly killing a wide range of microorganisms. We hypothesized that caths are expressed in mast cells (MCs), because these cells have been previously associated with inherent antimicrobial activity. Cultured murine MCs contained abundant amounts of cathelin-related antimicrobial peptide (AMP), the murine cath, and this expression was inducible by LPS or lipoteichoic acid. Human skin MCs also expressed cath as detected by immunohistochemical analysis for the human cath LL-37. The functional significance of this expression was shown by comparing MCs cultured from normal mice to MCs from littermates deficient in the cathelin-related AMP gene (Cnlp(-)). MCs derived from Cnlp(-/-) animals had a 50% reduction in their ability to kill group A STREPTOCOCCUS: These MCs expressed equivalent amounts of mRNA for murine beta-defensin-4, a beta-defensin AMP. Thus, different antimicrobials can be identified in MCs, and the presence of cath is necessary for efficient bacterial killing. These observations suggest that the presence of cath is vital to the ability of mammalian MCs to participate in antimicrobial defense.

It is widely accepted that unrepaired or misrepaired DNA double strand breaks (DSBs) lead to the formation of chromosome aberrations. DSBs induced in the DNA of higher eukaryotes by endogenous processes or exogenous agents can in principle be repaired either by non-homologous endjoining (NHEJ), or homology directed repair (HDR). The basis on which the selection of the DSB repair pathway is made remains unknown but may depend on the inducing agent, or process. Evaluation of the relative contribution of NHEJ and HDR specifically to the repair of ionizing radiation (IR) induced DSBs is important for our understanding of the mechanisms leading to chromosome aberration formation. Here, we review recent work from our laboratories contributing to this line of inquiry. Analysis of DSB rejoining in irradiated cells using pulsed-field gel electrophoresis reveals a fast component operating with half times of 10-30 min. This component of DSB rejoining is severely compromised in cells with mutations in DNA-PKcs, Ku, DNA ligase IV, or XRCC4, as well as after chemical inhibition of DNA-PK, indicating that it reflects classical NHEJ; we termed this form of DSB rejoining D-NHEJ to signify its dependence on DNA-PK. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DSBs using an alternative pathway operating with slower kinetics (half time 2-10 h). This alternative, slow pathway of DSB rejoining remains unaffected in mutants deficient in several genes of the RAD52 epistasis group, suggesting that it may not reflect HDR. We proposed that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway. Biochemical studies confirm the presence in cell extracts of DNA end joining activities operating in the absence of DNA-PK and indicate the dominant role for D-NHEJ, when active. These observations in aggregate suggest that NHEJ, operating via two complementary pathways, B-NHEJ and D-NHEJ, is the main mechanism through which IR-induced DSBs are removed from the DNA of higher eukaryotes. HDR is considered to either act on a small fraction of IR induced DSBs, or to engage in the repair process at a step after the initial end joining. We propose that high speed D-NHEJ is an evolutionary development in higher eukaryotes orchestrated around the newly evolved DNA-PKcs and pre-existing factors. It achieves within a few minutes restoration of chromosome integrity through an optimized synapsis mechanism operating by a sequence of protein-protein interactions in the context of chromatin and the nuclear matrix. As a consequence D-NHEJ mostly joins the correct DNA ends and suppresses the formation of chromosome aberrations, albeit, without ensuring restoration of DNA sequence around the break. B-NHEJ is likely to be an evolutionarily older pathway with less optimized synapsis mechanisms that rejoins DNA ends with kinetics of several hours. The slow kinetics and suboptimal synapsis mechanisms of B-NHEJ allow more time for exchanges through the joining of incorrect ends and cause the formation of chromosome aberrations in wild type and D-NHEJ mutant cells.

The smallpox vaccine consists of live vaccinia virus and is generally considered the gold standard of vaccines, since it is the only one that has led to the complete eradication of an infectious disease from the human population. Renewed fears that smallpox might be deliberately released in an act of bioterrorism have led to resurgence in the study of immunity and immunological memory to vaccinia virus and other poxviruses. Here we review our current understanding of memory T-cell, memory B-cell, and antibody responses to vaccinia and related poxviruses, both in animal models and human subjects. Of particular interest are recent advances in understanding protective immunity to poxviruses, quantifying immunological memory to the smallpox vaccine in humans, and identifying major vaccinia-specific T-cell and B-cell epitopes. In addition, potential mechanisms for maintenance of immunological memory are discussed.

The C2 domain acts as a membrane-targeting module in a diverse group of proteins including classical protein kinase Cs (PKCs), where it plays an essential role in activation via calcium-dependent interactions with phosphatidylserine. The three-dimensional structures of the Ca(2+)-bound forms of the PKCalpha-C2 domain both in the absence and presence of 1, 2-dicaproyl-sn-phosphatidyl-L-serine have now been determined by X-ray crystallography at 2.4 and 2.6 A resolution, respectively. In the structure of the C2 ternary complex, the glycerophosphoserine moiety of the phospholipid adopts a quasi-cyclic conformation, with the phosphoryl group directly coordinated to one of the Ca(2+) ions. Specific recognition of the phosphatidylserine is reinforced by additional hydrogen bonds and hydrophobic interactions with protein residues in the vicinity of the Ca(2+) binding region. The central feature of the PKCalpha-C2 domain structure is an eight-stranded, anti-parallel beta-barrel with a molecular topology and organization of the Ca(2+) binding region closely related to that found in PKCbeta-C2, although only two Ca(2+) ions have been located bound to the PKCalpha-C2 domain. The structural information provided by these results suggests a membrane binding mechanism of the PKCalpha-C2 domain in which calcium ions directly mediate the phosphatidylserine recognition while the calcium binding region 3 might penetrate into the phospholipid bilayer.

BCL6 is a transcriptional repressor required in mature B cells during the germinal center (GC) reaction. Multiple mechanisms act coordinately to timely modulate BCL6 expression at transcriptional and post-transcriptional levels. BCL6 prevents premature activation and differentiation of GC B cells and provides an environment tolerant of the DNA breaks associated with immunoglobulin gene remodeling mechanisms involved in the production of high-affinity antibodies of different isotypes. The critical functions exerted by BCL6 during normal B-cell development can be hijacked by the malignant transformation process. Indeed, BCL6 is targeted by genetic aberrations and acts as an oncogene in GC-derived lymphomas. The aberrations affecting BCL6 interfere with the multiple levels of regulation that grant a fine tuning of BCL6 expression and activity in physiologic conditions. This review summarizes the current knowledge on BCL6 function and its role in lymphomagenesis.

Mechanisms of signal transduction regulation remain a fundamental question in a variety of biological processes and diseases. Previous evidence indicates that the primary cilium can act as a signalling hub, but its exact role in many of the described pathways has remained elusive. Here, we investigate the mechanism of cilia-mediated regulation of the canonical Wnt pathway. We found that primary cilia dampen canonical Wnt signalling through a spatial mechanism involving compartmentalization of signalling components. The cilium, through regulated intraflagellar transport, diverts Jouberin (Jbn), a ciliopathy protein and context-specific Wnt pathway regulator, away from the nucleus and limits β-catenin nuclear entry. This repressive regulation does not silence the pathway, but instead maintains a discrete range of Wnt responsiveness; cells without cilia have potentiated Wnt responses, whereas cells with multiple cilia have inhibited responses. Furthermore, we show that this regulation occurs during embryonic development and is disrupted in cancer cell proliferation. Together these data explain a spatial mechanism of Wnt signalling regulation that may provide insight into ciliary regulation of other signalling pathways.