Cytotoxic T-lymphocyte activation involves a cascade of signalling and adhesion events.

In addition to the antigen-specific T-cell receptor (TCR), T cells bear an array of 'accessory' molecules that can contribute to stable adhesion to the antigen-bearing cell and provide costimulatory signals. For several of these, T-cell adhesion to the ligand can be activated by TCR-dependent signalling (a signal from the TCR primes the coreceptor to bind to its ligand). It is unclear whether the individual coreceptors share common mechanisms of priming and cosignalling, and perhaps act in a redundant manner, or whether they act in a distinct way and contribute uniquely to the activation process. We report here the use of isolated alloantigen, class I proteins and fibronectin ligands to show that coreceptors on cytotoxic T lymphocytes are activated sequentially and deliver distinct biochemical signals on binding to their ligands. TCR engagement activates CD8 by a protein tyrosine kinase-dependent pathway, and CD8 then acts as a signal for initiation of polyphosphoinositide hydrolysis on binding to class I. In contrast, activated adhesion to fibronectin does not initiate polyphosphoinositide hydrolysis, but amplifies hydrolysis once it has been initiated. Thus, cytotoxic T-lymphocyte activation involves a TCR-initiated cascade of adhesion and signalling events leading to response.     

Essential role for Gab2 in the allergic response.

Dos/Gab family scaffolding adapters (Dos, Gab1, Gab2) bind several signal relay molecules, including the protein-tyrosine phosphatase Shp-2 and phosphatidylinositol-3-OH kinase (PI(3)K); they are also implicated in growth factor, cytokine and antigen receptor signal transduction. Mice lacking Gab1 die during embryogenesis and show defective responses to several stimuli. Here we report that Gab2-/- mice are viable and generally healthy; however, the response (for example, degranulation and cytokine gene expression) of Gab2-/- mast cells to stimulation of the high affinity immunoglobulin-epsilon (IgE) receptor Fc(epsilon)RI is defective. Accordingly, allergic reactions such as passive cutaneous and systemic anaphylaxis are markedly impaired in Gab2-/- mice. Biochemical analyses reveal that signalling pathways dependent on PI(3)K, a critical component of Fc(epsilon)RI signalling, are defective in Gab2-/- mast cells. Our data identify Gab2 as the principal activator of PI(3)K in response to Fc(epsilon)RI activation, thereby providing genetic evidence that Dos/Gab family scaffolds regulate the PI(3)K pathway in vivo. Gab2 and/or its associated signalling molecules may be new targets for developing drugs to treat allergy.     

串联反应合成2-氨基-3,5二氰基-4H-吡喃衍生物

通过以丙二腈和氰基查尔酮作为原料,使用串联反应开发了一种2-氨基-3,5-二氰基-4H-吡喃衍生物的快速且高效的方法。筛选了催化剂、溶剂、反应时间、温度以及原料摩尔浓度对产率的影响。该方法具有产率高、反应条件温和以及催化剂廉价易得的优点。     

CoFe2O4/CoFe2S4活化PMS性能及机理

通过简单的两步水热法合成CoFe2 O4/CoFe2 S4复合物.扫描电子显微镜(SEM)、透射电子显微镜(TEM)、X射线衍射(XRD)及X射线光电子能谱(XPS)物性表征表明,CoFe2O4/CoFe2 S4复合物由纳米片和纳米花颗粒混杂构成.与CoFe2 O4相比,原位硫化引入轻微缺陷可以增加活性位点,从而促进CoFe2 O4/CoFe2 S4复合物的氧化还原活性,赋予CoFe2 O4/CoFe2 S4催化过一硫酸盐(PMS)体系更优异的氧化降解能力.进一步的参数优化实验表明,CoFe2 O4/CoFe2 S4与PMS体系氧化降解黄连素的最佳条件为1 mg/L CoFe2 O4/CoFe2 S4、0.8 mmol/L PMS、温度40℃、pH 7.0,该条件下黄连素的降解率在60 min内可达98.4％.CoFe2 O4/CoFe2 S4循环使用3次后,黄连素降解率依然在90％ 以上.电子顺磁共振(EPR)研究表明,CoFe2 O4/CoFe2 S4与PMS体系产生的主要活性氧化物种为SO4-和HO.自由基抑制实验证明,SO4-是降解黄连素的主要活性物种.     

Millisecond activation of transducin in the cyclic nucleotide cascade of vision

Cyclic GMP has been implicated as a messenger molecule involved in visual transduction. Photoexcited rhodopsin (R*) binds to a multisubunit membrane protein called transducin (T) and stimulates the exchange of a bound GDP molecule for GTP. This leads to the release of the alpha-subunit of T with bound GTP (T alpha-GTP), which activates a cyclic GMP phosphodiesterase. The question arises as to whether the hydrolysis of cyclic GMP that results from activation of the phosphodiesterase is sufficiently rapid to be involved in visual excitation, which occurs on a time scale of approximately 2 s in the single-photon limit. Previous studies have suggested that the cyclic GMP phosphodiesterase is activated in less than 100 ms at moderate light levels. We report here light scattering studies of magnetically orientated frog rod outer segments which show that a molecule of R* catalyses the activation of a molecule of T in about 1 ms. Thus, hundreds of molecules can be activated within the response time of vision in the single-photon limit, and the formation of T alpha-GTP is fast enough for it to be a key step in visual transduction.     

Dopamine activation of the arachidonic acid cascade as a basis for D1/D2 receptor synergism

Understanding the actions of the neurotransmitter dopamine in the brain is important in view of its roles in neuropsychiatric illnesses. Dopamine D1 receptors, which stimulate both adenylyl cyclase and phospholipase C, and D2 receptors, which inhibit them, can nevertheless act synergistically to produce many electrophysiological and behavioral responses. Because this functional synergism can occur at the level of single neurons, another, as yet unidentified, signalling pathway activated by dopamine has been hypothesized. We report here that in Chinese hamster ovary (CHO) cells transfected with the D2 receptor complementary DNA, D2 agonists potently enhanced arachidonic acid release, provided that such release has been initiated by stimulating constitutive purinergic receptors or by increasing intracellular Ca2+. In CHO cells expressed D1 receptors, D1 agonists exert no such effect. When D1 and D2 receptors are coexpressed, however, activation of both subtypes results in a marked synergistic potentiation of arachidonic acid release. The numerous actions of arachidonic acid and its metabolites in neuronal signal transduction suggest that facilitation of its release may be implicated in dopaminergic responses, such as feedback inhibition mediated by D2 autoreceptors, and may constitute a molecular basis for D1/D2 receptor synergism.     

Essential role for collectrin in renal amino acid transport

Angiotensin -converting enzyme 2 (ACE2) is a regulator of the renin angiotensin system involved in acute lung failure, cardiovascular functions and severe acute respiratory syndrome (SARS) infections in mammals. A gene encoding a homologue to ACE2, termed collectrin (Tmem27), has been identified in immediate proximity to the ace2 locus. The in vivo function of collectrin was unclear. Here we report that targeted disruption of collectrin in mice results in a severe defect in renal amino acid uptake owing to downregulation of apical amino acid transporters in the kidney. Collectrin associates with multiple apical transporters and defines a novel group of renal amino acid transporters. Expression of collectrin in Xenopus oocytes and Madin-Darby canine kidney (MDCK) cells enhances amino acid transport by the transporter B(0)AT1. These data identify collectrin as a key regulator of renal amino acid uptake.     

Essential role for oncogenic Ras in tumour maintenance.

Advanced malignancy in tumours represents the phenotypic endpoint of successive genetic lesions that affect the function and regulation of oncogenes and tumour-suppressor genes. The established tumour is maintained through complex and poorly understood host-tumour interactions that guide processes such as angiogenesis and immune sequestration. The many different genetic alterations that accompany tumour genesis raise questions as to whether experimental cancer-promoting mutations remain relevant during tumour maintenance. Here we show that melanoma genesis and maintenance are strictly dependent upon expression of H-RasV12G in a doxycycline-inducible H-Ras12G mouse melanoma model null for the tumour suppressor INK4a. Withdrawal of doxycycline and H-RasV12G down-regulation resulted in clinical and histological regression of primary and explanted tumours. The initial stages of regression involved marked apoptosis in the tumour cells and host-derived endothelial cells. Although the regulation of vascular endothelial growth factor (VEGF) was found to be Ras-dependent in vitro, the failure of persistent endogenous and enforced VEGF expression to sustain tumour viability indicates that the tumour-maintaining actions of activated Ras extend beyond the regulation of VEGF expression in vivo. Our results provide genetic evidence that H-RasV12G is important in both the genesis and maintenance of solid tumours.     

A role for Wnt signalling in self-renewal of haematopoietic stem cells

Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated beta-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.     

Essential role for Nix in autophagic maturation of erythroid cells

Erythroid cells undergo enucleation and the removal of organelles during terminal differentiation. Although autophagy has been suggested to mediate the elimination of organelles for erythroid maturation, the molecular mechanisms underlying this process remain undefined. Here we report a role for a Bcl-2 family member, Nix (also called Bnip3L), in the regulation of erythroid maturation through mitochondrial autophagy. Nix(-/-) mice developed anaemia with reduced mature erythrocytes and compensatory expansion of erythroid precursors. Erythrocytes in the peripheral blood of Nix(-/-) mice exhibited mitochondrial retention and reduced lifespan in vivo. Although the clearance of ribosomes proceeded normally in the absence of Nix, the entry of mitochondria into autophagosomes for clearance was defective. Deficiency in Nix inhibited the loss of mitochondrial membrane potential (DeltaPsi(m)), and treatment with uncoupling chemicals or a BH3 mimetic induced the loss of DeltaPsi(m) and restored the sequestration of mitochondria into autophagosomes in Nix(-/-) erythroid cells. These results suggest that Nix-dependent loss of DeltaPsi(m) is important for targeting the mitochondria into autophagosomes for clearance during erythroid maturation, and interference with this function impairs erythroid maturation and results in anaemia. Our study may also provide insights into molecular mechanisms underlying mitochondrial quality control involving mitochondrial autophagy.     

Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy

Evading apoptosis is considered to be a hallmark of cancer, because mutations in apoptotic regulators invariably accompany tumorigenesis. Many chemotherapeutic agents induce apoptosis, and so disruption of apoptosis during tumour evolution can promote drug resistance. For example, Akt is an apoptotic regulator that is activated in many cancers and may promote drug resistance in vitro. Nevertheless, how Akt disables apoptosis and its contribution to clinical drug resistance are unclear. Using a murine lymphoma model, we show that Akt promotes tumorigenesis and drug resistance by disrupting apoptosis, and that disruption of Akt signalling using the mTOR inhibitor rapamycin reverses chemoresistance in lymphomas expressing Akt, but not in those with other apoptotic defects. eIF4E, a translational regulator that acts downstream of Akt and mTOR, recapitulates Akt's action in tumorigenesis and drug resistance, but is unable to confer sensitivity to rapamycin and chemotherapy. These results establish Akt signalling through mTOR and eIF4E as an important mechanism of oncogenesis and drug resistance in vivo, and reveal how targeting apoptotic programmes can restore drug sensitivity in a genotype-dependent manner.     

Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour suppression

Reactive oxygen species are involved in many cellular metabolic and signalling processes and are thought to have a role in disease, particularly in carcinogenesis and ageing. We have generated mice with targeted inactivation of Prdx1, a member of the peroxiredoxin family of antioxidant enzymes. Here we show that mice lacking Prdx1 are viable and fertile but have a shortened lifespan owing to the development beginning at about 9 months of severe haemolytic anaemia and several malignant cancers, both of which are also observed at increased frequency in heterozygotes. The haemolytic anaemia is characterized by an increase in erythrocyte reactive oxygen species, leading to protein oxidation, haemoglobin instability, Heinz body formation and decreased erythrocyte lifespan. The malignancies include lymphomas, sarcomas and carcinomas, and are frequently associated with loss of Prdx1 expression in heterozygotes, which suggests that this protein functions as a tumour suppressor. Prdx1-deficient fibroblasts show decreased proliferation and increased sensitivity to oxidative DNA damage, whereas Prdx1-null mice have abnormalities in numbers, phenotype and function of natural killer cells. Our results implicate Prdx1 as an important defence against oxidants in ageing mice.     

Essential role for phosphatidylinositol 4,5-bisphosphate in yeast cell proliferation

The responses of mammalian cells to external signals are commonly mediated by intracellular secondary messengers, among which are the breakdown products of phosphatidylinositol 4,5-bisphosphate (PIP2): 1,2-diacylglycerol (DG) and inositol 1,4,5-triphosphate (IP3) (refs 1-7). Although phosphoinositide turnover in the yeast Saccharomyces cerevisiae has been shown to be regulated by glucose and sterol, as yet no definitive function has been ascribed to yeast phosphoinositides. We have recently developed a monoclonal antibody specific for PIP2 and reported that it inhibits mitogenesis of mammalian cells stimulated by platelet-derived growth factor and bombesin. We now report that when introduced into yeast cells by electroporation this antibody inhibits their growth. Furthermore, several yeast mutants with temperature-dependent growth defects are altered in their sensitivity to our antibody and are found to have specific alterations in their phosphoinositide metabolism.     

Essential role for the p110delta phosphoinositide 3-kinase in the allergic response

Inflammatory substances released by mast cells induce and maintain the allergic response. Mast cell differentiation and activation are regulated, respectively, by stem cell factor (SCF; also known as Kit ligand) and by allergen in complex with allergen-specific immunoglobulin E (IgE). Activated SCF receptors and high-affinity receptors for IgE (FcvarepsilonRI) engage phosphoinositide 3-kinases (PI(3)Ks) to generate intracellular lipid second messenger signals. Here, we report that genetic or pharmacological inactivation of the p110delta isoform of PI(3)K in mast cells leads to defective SCF-mediated in vitro proliferation, adhesion and migration, and to impaired allergen-IgE-induced degranulation and cytokine release. Inactivation of p110delta protects mice against anaphylactic allergic responses. These results identify p110delta as a new target for therapeutic intervention in allergy and mast-cell-related pathologies.     

The role of disparity-sensitive cortical neurons in signalling the direction of self-motion

Movement of an observer through the environment generates motion on the retina. This optic flow provides information about the direction of self-motion, but only if it contains differential motion of elements at different depths. If the observer tracks a stationary object while moving in a direction different from his line of sight, the images of objects in the foreground and in the background move in opposite directions. We have found neurons in the cerebral cortex of monkeys that prefer one direction of motion when the disparity of a stimulus corresponds to foreground motion and prefer the opposite direction when the disparity corresponds to background motion. We propose that these neurons contribute a signal about the direction of self-motion.