Non-steroidal anti-inflammatory drugs (NSAIDs) in Alzheimer's disease: old and new mechanisms of action

Alzheimer's disease (AD) is characterized by cerebral deposits of beta-amyloid (A beta) peptides and neurofibrillary tangles (NFT) which are surrounded by inflammatory cells. Epidemiological studies have shown that prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD and delays the onset of the disease. It has been postulated that some NSAIDs target pathological hallmarks of AD by interacting with several pathways, including the inhibition of cyclooxygenases (COX) and activation of the peroxisome proliferator-activated receptor gamma. A variety of experimental studies indicate that a subset of NSAIDs such as ibuprofen, flurbiprofen, indomethacin and sulindac also possess A beta-lowering properties in both AD transgenic mice and cell cultures of peripheral, glial and neuronal origin. While COX inhibition occurs at low concentrations in vitro (nM-low microm range), the A beta-lowering activity is observed at high concentrations (< or = 50 microm). Nonetheless, studies with flurbiprofen or ibuprofen in AD transgenic mice show that the effects on A beta levels or deposition are attained at plasma levels similar to those achieved in humans at therapeutic dosage. Still, it remains to be assessed whether adequate concentrations are reached in the brain. This is a crucial aspect that will allow defining the dose-window and the length of treatment in future clinical trials. Here, we will discuss how the combination of anti-amyloidogenic and anti-inflammatory activities of certain NSAIDs may produce a profile potentially relevant to their clinical use as disease-modifying agents for the treatment of AD.     

Inhibition of platelet-derived growth factor-mediated signal transduction by transforming ras. Suppression of receptor autophosphorylation.

The function of ras protein and its relationship to growth-factor mediated signal transduction remain unclear. The demonstration that the expression of transforming ras (v-ras) can block the stimulation of growth-related gene expression and cell division mediated by the platelet-derived growth factor (PDGF) may provide a model for the functional interation of ras with growth factor receptors. In the current studies, we have demonstrated that this blockade by v-ras of PDGF-BB signal transduction occurs very early in signal transduction, at the level of PDGF receptor autophosphorylation. Although the expression of PDGF receptor as detected by Western blot with anti-PDGF receptor antibody was not diminished in v-ras-transformed murine Balb/c 3T3 fibroblasts, the autophosphorylation of PDGF receptor in response to ligand (recombinant PDGF-BB homodimer) stimulation was profoundly suppressed. This same phenomenon of v-ras-mediated PDGF receptor autophosphorylation inhibition was also demonstrated in normal rat kidney fibroblasts. Further, factor(s) present in v-ras-expressing fibroblasts found in the membrane fractions of these cells can dominantly inhibit the autophosphorylation of the PDGF receptor obtained from normal fibroblasts. These findings suggest a role for ras in one of the earliest steps of the signal transduction pathway.     

Sphingosine-1-phosphate inhibits PDGF-induced chemotaxis of human arterial smooth muscle cells: spatial and temporal modulation of PDGF chemotactic signal transduction

Activation of the PDGF receptor on human arterial smooth muscle cells (SMC) induces migration and proliferation via separable signal transduction pathways. Sphingosine-1-phosphate (Sph-1-P) can be formed following PDGF receptor activation and therefore may be implicated in PDGF-receptor signal transduction. Here we show that Sph-1-P does not significantly affect PDGF-induced DNA synthesis, proliferation, or activation of mitogenic signal transduction pathways, such as the mitogen-activated protein (MAP) kinase cascade and PI 3-kinase, in human arterial SMC. On the other hand, Sph-1-P strongly mimics PDGF receptor-induced chemotactic signal transduction favoring actin filament disassembly. Although Sph-1-P mimics PDGF, exogenously added Sph-1-P induces more prolonged and quantitatively greater PIP2 hydrolysis compared to PDGF-BB, a markedly stronger calcium mobilization and a subsequent increase in cyclic AMP levels and activation of cAMP-dependent protein kinase. This excessive and prolonged signaling favors actin filament disassembly by Sph-1-P, and results in inhibition of actin nucleation, actin filament assembly and formation of focal adhesion sites. Sph-1-P-induced interference with the dynamics of PDGF-stimulated actin filament disassembly and assembly results in a marked inhibition of cell spreading, of extension of the leading lamellae toward PDGF, and of chemotaxis toward PDGF. The results suggest that spatial and temporal changes in phosphatidylinositol turnover, calcium mobilization and actin filament disassembly may be critical to PDGF-induced chemotaxis and suggest a possible role for endogenous Sph-1-P in the regulation of PDGF receptor chemotactic signal transduction.     

Overexpression and purification of human calcitonin gene-related peptide-receptor component protein in Escherichia coli

Calcitonin gene-related peptide (CGRP) is a neuropeptide secreted by the central and peripheral nervous system nerves that has important physiological functions such as vasodilation, cardiotonic actions, metabolic and pro-inflammatory effects. The CGRP receptor is unique among G-protein coupled receptors in that a functional CGRP receptor consists of at least three proteins: calcitonin like receptor (CLR), receptor activity modifying protein (RAMP1) and receptor component protein (RCP). RCP is a required factor in CGRP-mediated signal transduction and it couples the CGRP receptor to the signal transduction pathway. Here, we describe methods to overexpress and purify RCP for structure-function studies. Human RCP was cloned and overexpressed with a poly-histidine tag and as a maltose binding protein (MBP) fusion in Escherichia coli using commercially available expression vectors. While His tagged RCP is prone to aggregation, solubility is improved when RCP is expressed as a MBP fusion. Expression and purification procedures for these constructs are described. Results from these studies will facilitate structural analysis of human RCP, and allow further understanding of RCP function.     

The dynamics of protein phosphorylation in bacterial chemotaxis

The chemotaxis signal transduction pathway allows bacteria to respond to changes in concentration of specific chemicals (ligands) by modulating their swimming behavior. The pathway includes ligand binding receptors, and the CheA, CheY, CheW, and CheZ proteins. We showed previously that phosphorylation of CheY is activated in reactions containing receptor, CheW, CheA, and CheY. Here we demonstrate that this activation signal results from accelerated autophosphorylation of the CheA kinase. Evidence for a second signal transmitted by a ligand-bound receptor, which corresponds to inhibition of CheA autophosphorylation, is also presented. We postulate that CheA can exist in three forms: a "closed" form in the absence of receptor and CheW; an "open" form that results from activation of CheA by receptor and CheW; and a "sequestered" form in reactions containing ligand-bound receptor and CheW. The system's dynamics depends on the relative distribution of CheA among these three forms at any time.     

Dissociation of PTPase levels from their modulation of insulin receptor signal transduction

Protein tyrosine phosphatases have been implicated in the regulation of receptor tyrosine kinase signalling pathways, including that of the insulin receptor. Here, cell density-dependent changes in PTPase expression have been exploited to investigate the relationship between cellular PTPase levels and the insulin receptor signal transduction pathway. Increasing cell density (20%, 50%, and >90%) in the rat McA-RH7777 hepatoma cell line resulted in increased protein expression of the receptor-like PTPase LAR (14-fold), and the nonreceptor PTPases PTP1B (11-fold) and SHP2 (10-fold). Each of these PTPases has previously been implicated in regulating insulin receptor signal transduction. Despite these marked increases, maximum insulin receptor autophosphorylation as well as receptor expression actually increased 2-fold. MAP kinase also increased approximately 2-fold as a function of cell density and paralleled increases in expression levels. Neither sensitivity nor maximum responsiveness to insulin were decreased at increasing cell densities as assessed by activation of PI 3-kinase. Duration of response was also unimpaired. These results suggest that expression levels of relevant PTPases are not the primary determinant in their modulation of insulin receptor kinase activity. Restricted accessibility at the molecular level or involvement of accessory proteins may be more critical parameters.     

Analysis of the nonsteroidal anti-inflammatory drug literature for potential developmental toxicity in rats and rabbits

BACKGROUND: Nonsteroidal anti‐inflammatory drugs (NSAIDs) are among the most commonly prescribed to pregnant women. Some case‐control studies have linked the NSAIDs aspirin and indomethacin with a risk of congenital abnormalities and low birthweight. High doses of aspirin produce developmental toxicity in rats (e.g., gastroschisis/umbilical hernia, diaphragmatic hernia [DH]) when administered during sensitive windows of development. Unlike other NSAIDs, aspirin irreversibly inhibits cyclooxygenases (COXs) 1 and 2. Hence, the developmental toxicity seen in rats after exposure to aspirin may be due to the irreversible inhibition of COX‐1 and/or COX‐2. If so, other NSAIDs, which act through a reversible inhibition of COX, may produce a weak developmental toxicity signal or no developmental toxicity signal when tested in preclinical models. To investigate this relationship, a comprehensive analysis of the NSAID developmental toxicity literature was undertaken to determine whether NSAIDs other than aspirin induce developmental anomalies similar to those elicited by aspirin. METHODS: Developmental toxicity studies were identified through literature searches of PubMed and TOXNET, and pregnancy outcome data were extracted and tabulated. By using a set of defined criteria, each study was evaluated for quality and assigned to one of five tiers. The relation between certain malformations and NSAID treatment was analyzed for the best studies (tiers 1–4) by using concurrent control data (Mantel–Haenszel and permutation tests) and by combining the concurrent control data with historical control data (χ² test and permutation tests). RESULTS: A qualitative analysis of these data led to a focus on three types of malformations: DH, ventricular septal defects (VSDs), and midline defects (MDs). In rats, the incidences of VSD and MD were increased among fetuses treated with NSAIDs when compared with the concurrent controls. The extent of the increase was attenuated when the data from the aspirin studies were excluded from the analysis. There were no qualifying (i.e., tiers 1–4) aspirin studies conducted in rabbits, but the incidences of the three defects were increased over control incidences among non‐aspirin NSAID‐treated animals. Statistical analysis of these data was subsequently conducted. When tiers 1–4 were combined and compared with concurrent controls plus the most appropriate historical control database, the strongest associations were between NSAID treatment and VSD in rats, VSD in rabbits, and MD in rabbits. There also was some suggestion of an association between NSAID treatment and DH in rabbits. CONCLUSIONS: This analysis of the non‐clinical NSAID literature demonstrated a possible association between exposure to NSAIDs and developmental anomalies. The anomalies were similar for aspirin and for other NSAIDs, but effects occurred at a much lower incidence with non‐aspirin NSAIDs than previously reported with aspirin. Such a finding is consistent with the concept that reversible inhibition of COX‐1 and/or COX‐2 by other NSAIDs would produce weaker developmental toxicity signals than aspirin. However, there were limitations of the evaluated studies: (1) there were very few robust International Conference on Harmonization–compliant studies conducted with NSAIDs in the published literature; (2) many of the studies were conducted at doses well below the maximum tolerated dose (MTD), where effects are rarely seen; and (3) numerous studies were conducted above the MTD, where reduced numbers of fetuses hampered detection of low‐incidence findings. Although weak associations were observed, these limitations prevented us from definitively determining the presence or absence of a developmental toxicity signal from the existing body of NSAID data. Further exploration of this hypothesis will require assessing the potential association in animal models by using dose levels centered around the MTD. Birth Defects Research (Part B) 68:5–26, 2003. © 2003 Wiley‐Liss, Inc.     

cAMP regulation of early gene expression in signal transduction mutants of Dictyostelium

We have examined the regulation of three early developmentally regulated genes in Dictyostelium. Two of these genes (D2 and M3) are induced by pulses of cAMP and the other (K5) is repressed. Expression of these genes has been examined in a number of developmental mutants that are specifically blocked in various aspects of the signal transduction/cAMP relay system involved in aggregation and control of early development. The mutant strains include Synag mutants, which are blocked in receptor-mediated activation of adenylate cyclase and do not relay cAMP pulses; FrigidA mutants, which are blocked in receptor-mediated activation of both adenylate cyclase and the putative phosphoinositol bisphosphate (PIP2) turnover pathway and appear to be mutations in the gene encoding one of the G alpha protein subunits; and a StreamerF allele, which lacks cGMP-specific cGMP phosphodiesterase. From the analysis of the developmental expression of these genes under a variety of conditions in these mutant strains, we have drawn a number of conclusions concerning the modes of regulation of these genes. Full induction of D2 and M3 genes requires cAMP interaction with the cell surface receptor and an "oscillation" of the receptor between active and adapted forms. Induction of these genes does not require activation of the signal transduction pathway that leads to adenylate cyclase activation and cAMP relay, but does require activation of other receptor-mediated intracellular signal transduction pathways, possibly that involving PIP2 turnover. Likewise, repression of the K5 gene requires pulses of cAMP. Expression of this gene is insensitive to cAMP pulses in FrigidA mutants, suggesting that a signal transduction pathway is necessary for its repression. Results using the StreamerF mutant suggest that the rise in cGMP in response to cAMP/receptor interactions may not be directly related to control of the pulse-induced genes. In addition, we have examined the effect of caffeine, which M. Brenner and S.D. Thomas (1984, Dev. Biol., 101, 136-146) showed preferentially blocks the cAMP relay system by blocking receptor-mediated activation of adenylate cyclase. We show that in many of the mutants and in an axenic wild-type strain, caffeine causes the induction of pulse-induced gene expression to almost wild-type levels or in some cases to higher than wild-type levels. Our data suggest that caffeine works by activating some step in the signal transduction pathway that must lie downstream from both the receptor and at least one of the G proteins and thus has effects other than simply blocking the receptor-mediated cAMP relay system.     

The role of the CGRP-receptor component protein (RCP) in adrenomedullin receptor signal transduction.

G protein-coupled receptors are usually thought to act as monomer receptors that bind ligand and then interact with G proteins to initiate signal transduction. In this study we report an intracellular peripheral membrane protein named the calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) required for signal transduction at the G protein-coupled receptor for adrenomedullin. Cell lines were made that expressed an antisense construct of the RCP cDNA, and in these cells diminished RCP expression correlated with loss of adrenomedullin signal transduction. In contrast, loss of RCP did not diminish receptor density or affinity, therefore RCP does not appear to act as a chaperone protein. Instead, RCP represents a novel class of protein required to couple the adrenomedullin receptor to the cellular signal transduction pathway. A candidate adrenomedullin receptor named the calcitonin receptor-like receptor (CRLR) has been described, which forms high affinity adrenomedullin receptors when co-expressed with the accessory protein receptor-activity modifying protein 2 (RAMP2). RCP co-immunoprecipitated with CRLR and RAMP2, indicating that a functional adrenomedullin receptor is composed of at least three proteins: the ligand binding protein (CRLR), an accessory protein (RAMP2), and a coupling protein for signal transduction (RCP).     

Pharmacological and molecular basis for dopamine D-2 receptor diversity

This review will focus on the main lines of evidence that suggest the existence of multiple types of dopamine D-2 receptors. Dopamine D-2 receptors share structural elements suggesting that they belong to a gene superfamily classified as G-protein-coupled receptors and show an archetypical topology predicted to consist of seven putative transmembrane domains. Activation of D-2 receptors results in a variety of responses, including inhibition of cyclic AMP formation, inhibition of phosphoinositol turnover, increase of K-channel activity, and identified, nor has the possible hierarchy of these regulatory proteins in transforming the incoming signal into a change of second-messenger levels. A lot of experimental data support the hypothesis that there are multiple signal-processing pathways activated by dopamine through D-2-receptor stimulation. Recently, the identification of dopaminergic drugs that discriminate among the different transduction pathways and the isolation of distinct cDNAs encoding proteins that share binding profile indicative of D-2 receptors clearly indicate multiple forms of D-2 receptors. Pharmacologically, at least two distinct categories of dopamine D-2 receptors exist in rat pituitary. The first (D-2a) is insensitive to BHT 920 and coupled to inhibition of adenylyl cyclase activity; the second (D-2b) is activated by BHT 920 and linked to voltage-dependent K channels. The two types of dopamine D-2 receptors differ in their structure, G-protein-coupled and effector. Each of the three basic receptor units shows a certain degree of heterogeneity, which may affect the quality and the kinetic of the response. This variety may represent the molecular basis for the diversity in pharmacological and functional profiles of different dopamine D-2 receptors located in various brain areas and peripheral tissues.     

Involvement of JAK-family tyrosine kinases in hematopoietin receptor signal transduction

A variety of cytokines, hormones and hematopoietic growth factors signal through the hematopoietin family of membrane receptors, which share several structural features, including a Trp-Ser-X-Trp-Ser motif and four paired cysteine residues. The signal transduction mechanisms utilized by these receptors have remained elusive, although tyrosine kinase activation has been one common element. Recently, a role for the cytoplasmic tyrosine kinases of the Janus kinase (JAK) family has been implicated in signalling by these receptors. There are currently three known JAK family kinases, including JAK1, JAK2 and TYK2. This review will focus on the role of such tyrosine kinases in hematopoietin receptor signal transduction, and address the possibility of the involvement also of unidentified Janus kinases.     

Effects of testosterone on fat cell lipolysis. Species differences and possible role in polycystic ovarian syndrome

Testosterone is a potent regulator of lipolysis by influencing catecholamine signal transduction in fat cells. Major species differences exist as regards the testosterone effect. In rodents testosterone increases beta-adrenergic receptor mediated signals to lipolysis at multiple steps in the lipolytic cascade. The sex hormone also increases alpha2-adrenoceptor antilipolytic signalling in hamster which unlike rat express this receptor in their fat cells. In humans the region of adipose tissue is critical. Visceral fat cell lipolysis is not responsive to testosterone but this sex hormone decreases catecholamine-induced lipolysis in subcutaneous fat cells due to inhibition of the expression of beta2-adrenoceptors and hormone sensitive lipase. In polycystic ovarian syndrome (PCOS), which is characterized as a hyperandrogenic state, the lipolytic effect of catecholamine is decreased in subcutaneous adipocytes due to low content of beta2-adrenoceptors and hormone sensitive lipase. It is possible that the increased testosterone levels are responsible for these abnormalities in catecholamine signal transduction in subcutaneous fat cells of PCOS women. However, in visceral fat cells of PCOS women catecholamine-induced lipolysis is enhanced which cannot be explained by testosterone.     

Regulation of NF-κB-dependent gene expression by ligand-induced endocytosis of the interleukin-1 receptor

Interleukin-1 (IL-1) induces the internalization of its cognate receptor from the plasma membrane. However, it has remained elusive as to how this mechanism affects the IL-1-induced signal transduction. In this study, we used small-molecule inhibitors of receptor endocytosis to analyze the effects on IL-1-induced signal transduction pathways. We demonstrate that the inhibition of endocytosis down-modulates IL-1-induced NF-κB-dependent gene expression at a level downstream of nuclear translocation and DNA binding of NF-κB. Moreover, we report that the reduced NF-κB-dependent gene expression disrupts feedback inhibition loops terminating the activation of mitogen-activated protein kinases and down-regulating the expression of IL-1-induced mRNAs. Collectively, we show that the inhibition of endocytosis causes a dysregulation of IL-1-induced signal transduction and gene expression demonstrating an important role for receptor internalization in IL-1 signaling.     

Toll样受体4信号转导通路的干预

Toll样受体4(Toll like receptor 4,TLR4)是广泛表达于哺乳动物的跨膜受体,由于TLR4在人体的高表达与各种炎症反应相关联,抑制过高的TLR4表达可能是控制机体炎症损伤的新途径.目前的研究主要是针对TLR4的直接阻断与对TLR4的信号转导通路的抑制.由于TLR4的信号转导通路已经较为明确,从而研究对TLR4信号转导通路的抑制可能会对机体过强的炎症反应及损伤的控制产生有益作用.本文就当前针对抑制TLR4信号转导通路的研究作一综述.     

Natriuretic peptide receptor-C signaling and regulation

The natriuretic peptides (NP) are a family of three polypeptide hormones termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP regulates a variety of physiological parameters by interacting with its receptors present on the plasma membrane. These are of three subtypes NPR-A, NPR-B, and NPR-C. NPR-A and NPR-B are guanylyl cyclase receptors, whereas NPR-C is non-guanylyl cyclase receptor and is coupled to adenylyl cyclase inhibition or phospholipase C activation through inhibitory guanine nucleotide regulatory protein (Gi). ANP, BNP, CNP, as well as C-ANP(4-23), a ring deleted peptide that specifically interacts with NPR-C receptor inhibit adenylyl cyclase activity through Gi protein. Unlike other G-protein-coupled receptors, NPR-C receptors have a single transmembrane domain and a short cytoplasmic domain of 37 amino acids, which has a structural specificity like those of other single transmembrane domain receptors. A 37 amino acid cytoplasmic peptide is sufficient to inhibit adenylyl cyclase activity with an apparent Ki similar to that of ANP(99-126) or C-ANP(4-23). In addition, C-ANP(4-23) also stimulates phosphatidyl inositol (PI) turnover in vascular smooth muscle cells (VSMC) which is attenuated by dbcAMP and cAMP-stimulatory agonists, suggesting that NPR-C receptor-mediated inhibition of adenylyl cyclase and resultant decreased levels of cAMP may be responsible for NPR-C-mediated stimulation of PI turnover. Furthermore, the activation of NPR-C receptor by C-ANP(4-23) and CNP inhibits the mitogen-activated protein kinase activity stimulated by endothelin-3, platelet-derived growth factor, phorbol-12 myristate 13-acetate, suggesting that NPR-C receptor might also be coupled to other signal transduction system or that there may be an interaction of the NPR-C receptor and some other signaling pathways. In this review article, NPR-C receptor coupling to different signaling pathways and their regulation will be discussed.     

水稻穗发育功能基因对穗期氮肥的响应

为探讨氮肥对水稻(Oryza sativa)穗发育的调控作用, 使用高通量测序技术检测氮肥处理前后水稻叶片和幼穗组织中转录组的变化, 并从中筛选到大量差异表达基因。这些基因的功能涉及转录调控、激素代谢和信号转导、物质代谢和转运、胁迫响应、信号转导(受体)和蛋白质降解等。同时对目前克隆得到的穗发育相关基因进行分析, 发现在氮素穗肥的作用下, 部分重要功能基因的表达量发生了明显变化, 其中一些基因还参与调控水稻株高、抽穗期、分蘖和结实率等性状。对这些差异表达基因的功能研究有助于揭示氮素穗肥调控水稻每穗颖花数的分子机制。     

实夜蛾属和铃夜蛾属昆虫性信息素通讯系统的研究进展

实夜蛾属Heliothis和铃夜蛾属Helicoverpa昆虫的性信息素通讯系统主要包括雌蛾的性信息素合成和雄蛾对性信息素接收两个方面,每方面都有分子、细胞、系统水平上进行协同作用的生物过程。性信息素生物合成激活肽（PBAN）与其受体作用,启动信号转导系统,从而激活合成性信息素的酶系统来合成性信息素,利用化学和生物测定的方法鉴定出具有诱蛾活性的性信息素腺体组分及行为功能;性信息素分子与性信息素结合蛋白（PBP）的复合体同受体相互作用,启动信号转导系统,诱导产生神经信号,从而引起一系列性行为反应。这些生物过程受到各种内部和外部因素的影响。