Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor

Endothelin-1 was initially identified as a 21-residue potent vasoconstrictor peptide produced by vascular endothelial cells, but was subsequently found to have many effects on both vascular and non-vascular tissues. The discovery of three isopeptides of the endothelin family, ET-1, ET-2 and ET-3, each possessing a diverse set of pharmacological activities of different potency, suggested the existence of several different endothelin receptor subtypes. Endothelins may elicit biological responses by various signal-transduction mechanisms, including the G protein-coupled activation of phospholipase C and the activation of voltage-dependent Ca2+ channels. Thus, different subtypes of the endothelin receptor may use different signal-transduction mechanisms. Here we report the cloning of a complementary DNA encoding one subtype belonging to the superfamily of G protein-coupled receptors. COS-7 cells transfected with the cDNA express specific and high-affinity binding sites for endothelins, responding to binding by the production of inositol phosphates and a transient increase in the concentration of intracellular free Ca2+. The three endothelin isopeptides are roughly equipotent in displacing 125I-labelled ET-1 binding and causing Ca2+ mobilization. A messenger RNA corresponding to the cDNA is detected in many rat tissues including the brain, kidney and lung but not in vascular smooth muscle cells. These results indicate that this cDNA encodes a 'nonselective' subtype of the receptor which is different from the vascular smooth muscle receptor.     

Extensive proliferation of mature connective-tissue type mast cells in vitro

There are two phenotypically distinct subpopulations of mast cells in rodents: connective tissue-type mast cells (CTMC) and mucosal mast cells (MMC). These populations differ in their location, cell size, staining characteristics, ultrastructure, mediator content and T-cell dependency. Several investigators recently reported a further subclass of mast cells which arise when normal mouse haematopoietic cells are cultured with interleukin-3 (IL-3); IL-3 is an activity similar or identical to mast-cell growth factor, histamine-producing factor, or P-cell stimulating factor. These cultured mast cells are in many ways similar to MMC; they stain with Alcian blue but not safranin, contain chondroitin sulphate E proteoglycan rather than heparin proteoglycan and have relatively low histamine content, as do MMC. Although proliferation of MMC is known to be T-cell dependent in vivo and thought to be IL-3-dependent in vitro, the factors on which CTMC proliferation depends remain elusive. Here we show that mature CTMC purified from mouse peritoneal cells can proliferate in vitro in methylcellulose culture and maintain the appearance and function of CTMC. We also present evidence that mature CTMC cannot proliferate in the presence of pure IL-3 alone.     

Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease

Activation of naive CD4(+) T-helper cells results in the development of at least two distinct effector populations, Th1 and Th2 cells. Th1 cells produce cytokines (interferon (IFN)-gamma, interleukin (IL)-2, tumour-necrosis factor (TNF)-alpha and lymphotoxin) that are commonly associated with cell-mediated immune responses against intracellular pathogens, delayed-type hypersensitivity reactions, and induction of organ-specific autoimmune diseases. Th2 cells produce cytokines (IL-4, IL-10 and IL-13) that are crucial for control of extracellular helminthic infections and promote atopic and allergic diseases. Although much is known about the functions of these two subsets of T-helper cells, there are few known surface molecules that distinguish between them. We report here the identification and characterization of a transmembrane protein, Tim-3, which contains an immunoglobulin and a mucin-like domain and is expressed on differentiated Th1 cells. In vivo administration of antibody to Tim-3 enhances the clinical and pathological severity of experimental autoimmune encephalomyelitis (EAE), a Th1-dependent autoimmune disease, and increases the number and activation level of macrophages. Tim-3 may have an important role in the induction of autoimmune diseases by regulating macrophage activation and/or function.     

A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence

Plants sense potential microbial invaders by using pattern-recognition receptors to recognize pathogen-associated molecular patterns (PAMPs). In Arabidopsis thaliana, the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2) (ref. 2) and elongation factor Tu receptor (EFR) (ref. 3) act as pattern-recognition receptors for the bacterial PAMPs flagellin and elongation factor Tu (EF-Tu) (ref. 5) and contribute to resistance against bacterial pathogens. Little is known about the molecular mechanisms that link receptor activation to intracellular signal transduction. Here we show that BAK1 (BRI1-associated receptor kinase 1), a leucine-rich repeat receptor-like kinase that has been reported to regulate the brassinosteroid receptor BRI1 (refs 6,7), is involved in signalling by FLS2 and EFR. Plants carrying bak1 mutations show normal flagellin binding but abnormal early and late flagellin-triggered responses, indicating that BAK1 acts as a positive regulator in signalling. The bak1-mutant plants also show a reduction in early, but not late, EF-Tu-triggered responses. The decrease in responses to PAMPs is not due to reduced sensitivity to brassinosteroids. We provide evidence that FLS2 and BAK1 form a complex in vivo, in a specific ligand-dependent manner, within the first minutes of stimulation with flagellin. Thus, BAK1 is not only associated with developmental regulation through the plant hormone receptor BRI1 (refs 6,7), but also has a functional role in PRR-dependent signalling, which initiates innate immunity.     

Phorbol ester and diacylglycerol induce protein phosphorylation at tyrosine

The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is an efficient tumour promoter in vivo. In vitro, TPA activates the phospholipid- and Ca2+-dependent protein kinase, kinase C. This activation is believed to reflect the structural similarity between TPA and diacylglycerol, the endogenous protein kinase C activator which is produced in vivo by hydrolysis of phosphatidylinositol (reviewed in ref. 3). Protein kinase C phosphorylates protein substrates at serine and threonine residues in vitro. The effects of TPA on cultured fibroblasts--including enhanced hexose uptake, disruption of actin stress fibres and growth stimulation--are very similar to those induced by certain retrovirus transforming proteins and by peptide growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and multiplication-stimulating activity (MSA). These transforming proteins and mitogenic agents seem to act by inducing tyrosine-specific protein phosphorylation. Such observations suggested that some of the effects of TPA in vivo may be mediated by protein phosphorylation at tyrosine residues. A 42,000-molecular weight (42 K) polypeptide was previously shown to be phosphorylated at tyrosine in cells transformed by avian sarcoma viruses and in cells stimulated by EGF, PDGF or MSA (J. Cooper, personal communication and refs 11 and 12; this polypeptide was originally designated 43 K or spot n in ref. 10). We show here that this polypeptide also becomes phosphorylated at tyrosine in cells treated with TPA. Furthermore, exogenously added diacylglycerol likewise stimulates the phosphorylation of this protein at tyrosine.     

银杏叶提取物与人参皂苷对血管活性物质的影响

银杏叶提取物(GBE)和人参皂苷(GS)均具有显著的心血管药理活性,但未见二者配伍应用于心血管疾病治疗的研究和报道.采用血清药理学方法研究GBE与GS单独和配伍对氧化损伤内皮细胞代谢一氧化氮(NO)、内皮素(ET-1)及丙二醛(MDA)的影响.GBE、GS均可升高氧化损伤内皮细胞NO的代谢水平,降低其ET-1和MDA的代谢水平.二者配伍后抗脂质过氧化的作用增强,但对氧化损伤内皮细胞NO代谢的调节作用不及GBE和GS,分别降低了29.9%和66.7%,差异显著(P＜0.05);对ET-1代谢的调节作用虽优于GBE,但与GS相比降低了11.6%.这提示GBE和GS配伍后对内皮细胞血管活性物质NO和ET-1的调节作用未起到协同效果,GBE和GS配伍能否应用于心血管疾病的治疗还是一个值得探讨的课题.     

Transformation of cell adhesion properties by exogenously introduced E-cadherin cDNA

E-cadherin is a cell surface glycoprotein responsible for Ca2+-dependent intercellular adhesion between epithelial cells; it is also called uvomorulin, L-CAM (ref. 3), cell-CAM 120/80 (ref.4) or Arc-1 (ref. 5). Because blocking the action of E-cadherin by monoclonal antibodies causes dispersion of compact cell colonies, this molecule is thought to be an important factor for maintenance of multicellular systems. To demonstrate directly that E-cadherin is involved in cell-cell adhesion, we cloned full-length cDNA encoding E-cadherin from F9 cells and introduced it into L fibroblasts deficient in E-cadherin. These L cells acquire strong Ca2+-dependent aggregating activity by expressing the E-cadherin derived from the introduced cDNA and were morphologically transformed so as to form colonies in which cells were tightly connected to each other.     

Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck

The CD4 T-cell surface antigen is an integral membrane glycoprotein of relative molecular mass 55,000 which binds class II major histocompatibility complex (MHC) molecules expressed on antigen presenting cells (APCs). It is thought to stabilize physical interactions between T cells and APCs (for a review, see ref. 1). Evidence is accumulating that suggests that CD4 can transduce an independent signal during T-cell activation. It has recently been shown that CD4 expressed on human and murine T cells is physically associated with the Src-related tyrosine protein kinase p56lck (refs 7, 8). These results indicate that CD4 can function as a signal transducer and suggest that tyrosine phosphorylation events may be important in CD4-mediated signalling. Here, we present evidence that cross-linking of the CD4 receptor induces a rapid increase in the tyrosine-specific protein kinase activity of p56lck and is associated with the rapid phosphorylation of one of the subunits (zeta) of the T-cell receptor complex on tyrosine residues. These data provide direct evidence for a specific CD4 signal transduction pathway that is mediated through p56lck and suggest that some of the tyrosine phosphorylation events detected during antigen-mediated T-cell activation may result from signalling through this surface molecule.     

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.     

高压氧对慢性充血性心力衰竭患者血循环内皮细胞及脑钠素的影响

目的探讨充血性慢性心力衰竭(CHF)时血循环内皮细胞记数(CEC)、血浆脑钠素(BNP)、血浆内皮素(ET-1)及6酮前列腺素F1a(6-Keto-PGF1a)水平的变化及高压氧(HBO)治疗的干预效应.方法 60例CHF患者随机分为BBO治疗组30例,对照组30例,HBO组治疗压力0.2 MPa,1次/d,每周6次,共治疗4周,并检测用药前后自身外周血循环CEC数量、血浆ET-1,BNP及6-Keto-PGF1a的含量,30例CHF患者未行HBO治疗为对照组.结果高压氧治疗可使CEC数量、血浆ET-1及BNP水平明显降低及6-Keto-PGF1含量明显增加(P<0.01).结论慢性心力衰竭时存在血管内皮细胞受损,而高压氧治疗具有保护血管内皮细胞及改善心功能的作用.     

Retrovirus-induced de novo methylation of flanking host sequences correlates with gene inactivity

The pattern of DNA methylation changes during development of eukaryotes, and hypomethylation frequently correlates with gene expression (for reviews see refs 1-4). A causal relationship between hypermethylation and gene inactivity has been established for retroviral genomes which are methylated de novo when inserted into the germ line of mice (ref. 5; for review, see ref. 6). The mutual interaction of the provirus with the host genome can influence virus expression and can result in inactivation of the host gene by insertional mutagenesis. We report here that the insertion of a provirus can change the methylation pattern of the host DNA. Sequences flanking the provirus become methylated de novo within 1 kilobase (kb) of the integration site. In Mov-13 mice, which carry a lethal mutation of the alpha 1(I) collagen gene, de novo methylation of host DNA is associated with a change in chromatin conformation. This suggests that virus-induced DNA methylation can alter DNA-protein interactions and thereby interfere with correct gene activation during embryonic development.     

Non-canonical inflammasome activation targets caspase-11

Caspase-1 activation by inflammasome scaffolds comprised of intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and the adaptor ASC is believed to be essential for production of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 during the innate immune response. Here we show, with C57BL/6 Casp11 gene-targeted mice, that caspase-11 (also known as caspase-4) is critical for caspase-1 activation and IL-1β production in macrophages infected with Escherichia coli, Citrobacter rodentium or Vibrio cholerae. Strain 129 mice, like Casp11(-/-) mice, exhibited defects in IL-1β production and harboured a mutation in the Casp11 locus that attenuated caspase-11 expression. This finding is important because published targeting of the Casp1 gene was done using strain 129 embryonic stem cells. Casp1 and Casp11 are too close in the genome to be segregated by recombination; consequently, the published Casp1(-/-) mice lack both caspase-11 and caspase-1. Interestingly, Casp11(-/-) macrophages secreted IL-1β normally in response to ATP and monosodium urate, indicating that caspase-11 is engaged by a non-canonical inflammasome. Casp1(-/-)Casp11(129mt/129mt) macrophages expressing caspase-11 from a C57BL/6 bacterial artificial chromosome transgene failed to secrete IL-1β regardless of stimulus, confirming an essential role for caspase-1 in IL-1β production. Caspase-11 rather than caspase-1, however, was required for non-canonical inflammasome-triggered macrophage cell death, indicating that caspase-11 orchestrates both caspase-1-dependent and -independent outputs. Caspase-1 activation by non-canonical stimuli required NLRP3 and ASC, but caspase-11 processing and cell death did not, implying that there is a distinct activator of caspase-11. Lastly, loss of caspase-11 rather than caspase-1 protected mice from a lethal dose of lipopolysaccharide. These data highlight a unique pro-inflammatory role for caspase-11 in the innate immune response to clinically significant bacterial infections.     

Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery

Polo-like kinase-1 (PLK1) is an essential mitotic kinase regulating multiple aspects of the cell division process. Activation of PLK1 requires phosphorylation of a conserved threonine residue (Thr 210) in the T-loop of the PLK1 kinase domain, but the kinase responsible for this has not yet been affirmatively identified. Here we show that in human cells PLK1 activation occurs several hours before entry into mitosis, and requires aurora A (AURKA, also known as STK6)-dependent phosphorylation of Thr 210. We find that aurora A can directly phosphorylate PLK1 on Thr 210, and that activity of aurora A towards PLK1 is greatly enhanced by Bora (also known as C13orf34 and FLJ22624), a known cofactor for aurora A (ref. 7). We show that Bora/aurora-A-dependent phosphorylation is a prerequisite for PLK1 to promote mitotic entry after a checkpoint-dependent arrest. Importantly, expression of a PLK1-T210D phospho-mimicking mutant partially overcomes the requirement for aurora A in checkpoint recovery. Taken together, these data demonstrate that the initial activation of PLK1 is a primary function of aurora A.     

Acquisition of a multifunctional IgA+ plasma cell phenotype in the gut

The largest mucosal surface in the body is in the gastrointestinal tract, a location that is heavily colonized by microbes that are normally harmless. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the gastrointestinal tract is the production and transepithelial transport of poly-reactive IgA (ref. 1). Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T-cell help, undergo class switch recombination of their immunoglobulin receptor to IgA, and differentiate to become plasma cells. However, IgA-secreting plasma cells probably have additional attributes that are needed for coping with the tremendous bacterial load in the gastrointestinal tract. Here we report that mouse IgA(+) plasma cells also produce the antimicrobial mediators tumour-necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS), and express many molecules that are commonly associated with monocyte/granulocytic cell types. The development of iNOS-producing IgA(+) plasma cells can be recapitulated in vitro in the presence of gut stroma, and the acquisition of this multifunctional phenotype in vivo and in vitro relies on microbial co-stimulation. Deletion of TNF-α and iNOS in B-lineage cells resulted in a reduction in IgA production, altered diversification of the gut microbiota and poor clearance of a gut-tropic pathogen. These findings reveal a novel adaptation to maintaining homeostasis in the gut, and extend the repertoire of protective responses exhibited by some B-lineage cells.     

Histamine regulates T-cell and antibody responses by differential expression of H1 and H2 receptors

Many pathological processes, including those causing allergies and autoimmune diseases, are associated with the presence of specialized subsets of T helper cells (TH1 and TH2) at the site of inflammation. The diversity of TH1 and TH2 function is not predetermined but depends on signals that drive the cells towards either subset. Histamine, released from effector cells (mast cells and basophils) during inflammatory reactions can influence immune response. Here we report that histamine enhances TH1-type responses by triggering the histamine receptor type 1 (H1R), whereas both TH1- and TH2-type responses are negatively regulated by H2R through the activation of different biochemical intracellular signals. In mice, deletion of H1R results in suppression of interferon (IFN)-gamma and dominant secretion of TH2 cytokines (interleukin (IL)-4 and IL-13). Mutant mice lacking H2R showed upregulation of both TH1 and TH2 cytokines. Relevant to T-cell cytokine profiles, mice lacking H1R displayed increased specific antibody response with increased immunoglobulin-epsilon (IgE) and IgG1, IgG2b and IgG3 compared with mice lacking H2R. These findings account for an important regulatory mechanism in the control of inflammatory functions through effector-cell-derived histamine.