血小板活化因子抑制T细胞早期活化信号的转导

血小板活化因子 (PAF)是一种脂类介质 ,可由多种细胞分泌。PAF除能引起血小板凝集外 ,还能活化嗜中性粒细胞、嗜酸性粒细胞、单核细胞和巨噬细胞。PAF对T细胞的活化亦有调节作用 ,例如 ,PAF可抑制由PHA或CD3McAb诱导的T细胞增殖、IL 2和IL 4的合成、IL 2R(CD2 5 )的表达[1] 。本实验通过分别观察PAF对CD3McAb和PMA/iono mycin诱导的T细胞活化的作用 ,研究PAF对淋巴细胞活化的抑制作用是否与其对早期信号转导过程的调节有关。材料和方法淋巴细胞的制备和培养 :利用Ficoll Hypaque(Biochrom)密度梯度离心技术和E花结形成…     

血小板活化因子抗体的研制

血小板活化因子（ＰＡＦ）自发现以来,一直受到广泛的重视,现认为ＰＡＦ是一种具有激素样广泛生物学活性的物质［１］。关于血小板活化因子抗体的研制,国外有部分实验室有报道,国内少见报道,本文根据血小板活化因子特殊的化学结构,采用２种不同的方法制备出ＰＡＦ免疫原,经皮下多点和足底加淋巴结２种不同途径注射免疫新西兰白兔和Ｂａｌｂｃ小鼠,制备出效价较高、特异性较强的ＰＡＦ多克隆抗体。１　材料与方法１．１　材料　ＰＡＦ纯品、卵磷脂、胆固醇、弗氏完全佐剂（ＣＦＡ）、甲基化牛血清白蛋白（ＭＢＳＡ）均购自美国Ｓｉ…     

血小板活化因子与局部微循环障碍

血小板活化因子 (ＰＡＦ)是一种重要的血管活性物质 ,在局部微循环障碍的病理改变过程中起重要作用。１ＰＡＦ的代谢 :ＰＡＦ由白细胞、血小板及微血管内皮细胞等产生。ＰＡＦ是磷脂类物质 ,除由膜磷脂生成以外 ,细胞脂蛋白和血浆脂蛋白在分解时也可产生。ＰＡＦ被ＰＡＦ乙酰水解酶灭活。研究表明 ,栓塞和胎儿发育情况等可通过影响ＰＡＦ乙酰水解酶的活性 ,从而影响ＰＡＦ的含量及其微血管效应。Ｏｈｓｈｉｇｅ等观察了胎儿和孕妇的血浆ＰＡＦ乙酰水解酶活性 ,发现发育正常胎儿脐静脉的血浆ＰＡＦ乙酰水解酶活性显著低于母亲血浆 (Ｐ＜０．…     

血小板活化因子及其受体与淋巴细胞

血小板活化因子 (plateletactivatingfactor,PAF)是一种具有强大生物活性的磷脂类介质 ,在生物体内与其特异性受体结合而发挥作用。本文主要综述了PAF对淋巴细胞功能的影响及相关作用机制的研究进展。B淋巴细胞及大多数B细胞株上存在组成性PAF受体 ,受到刺激后还可诱生性表达 ;PAF能直接促进B淋巴细胞免疫球蛋白的分泌 ;并能抑制B淋巴细胞的凋亡。T淋巴细胞及大多数T淋巴细胞株上没有PAF受体表达 ,一般不产生和代谢PAF ;PAF能引起T淋巴细胞的趋化反应。PAF参与NK细胞的趋化反应及杀伤肿瘤细胞。     

087 血小板活化因子及其受体与淋巴细胞

血小板活化因子(platelet activating factor，PAF)是一种具有强大生物活性的磷脂类介质，在生物体内与其特异性受体结合而发挥作用。本文主要综述了PAF对淋巴细胞功能的影响及相关作用机制的研究进展。B淋巴细胞及大多数B细胞株上存在组成性PAF受体，受到刺激后还可诱生性表达；PAF能直接促进B淋巴细胞免疫球蛋白的分泌；并能抑制B淋巴细胞的凋亡。T淋巴细胞及大多数T淋巴细胞株上没有PAF受体表达，一般不产生和代谢PAF；PAF能引起T淋巴细胞的趋化反应。PAF参与NK细胞的趋化反应及杀伤肿瘤细胞。     

血小板活化因子对嗜酸性粒细胞的活化作用

用多粘菌素Ｂ注射，造成豚鼠腹腔渗出液中嗜酸性粒细胞（Ｅｏｓ）增多，分离出其中的Ｅｏｓ，测定血小板活化因子（ＰＡＦ）对Ｅｏｓ过氧化物酶（ＥＰＯ）活性、Ｅｏｓ沉淀密度和存活时间的作用。结果表明：ＰＡＦ能增强ＥＰＯ活性，诱导Ｅｏｓ脱颗粒，其最大作用浓度分别为１０＾－７ｍｏｌ／Ｌ和１０＾－６ｍｏｌ／Ｌ。淋巴细胞培养上清液能显著增强ＰＡＦ的脱颗粒作用。１０＾－６ｍｏｌ／Ｌ ＰＡＦ还能使１５．９％的正常密度Ｅ     

血小板活化因子及其受体与淋巴细胞

血小板活化因子（platelet activating factor,PAF)是一种具有强大生物活性的磷脂类介质，在生物体内与其特异性受体结合而发挥作用。本文主要综述了PAF对淋巴细胞功能的影响及相关作用机制的研究进展。B淋巴细胞及大多数B细胞株上存在组成性PAF受体，受到刺激后还可诱生性表达；PAF能直接促进B淋巴细胞免疫球蛋白的分泌；并能抑制B淋巴细胞的凋亡。T淋巴细胞及大多数T淋巴细胞 株上没有PAF受体表达，一般不产生和代谢PAF；PAF能引起T淋巴细胞的趋化反应。PAF参与NK细胞的趋化反应及杀伤肿瘤细胞。     

血小板活化因子介导肾小球系膜细胞产生活性氧的研究

为探讨血小板活化因子（ＰＡＦ）对肾小球系膜细胞（ＧＭＣ）产生活性氧的调节作用，本研究观察了ＰＡＦ对体外培养的大鼠ＧＭＣ产生超氧化物阴离子（Ｏ）和过氧化氢（Ｈ－２Ｏ－２）的影响。结果表明１０￣（－９）Ｍ／Ｌ的ＰＡＦ已能诱导ＧＭＣ产生Ｏ和Ｈ２Ｏ２（１．１４±０．４２ｎｍｏｌ／１０￣５ＧＭＣ、０．９７±０．１６ｎｍｏｌ／１０－５ＧＭＣ），这一效应呈剂量依赖性；溶ＰＡＦ无诱导ＧＭＣ产生活性氧作用；特异性ＰＡＦ受体拮抗剂ＢＮ５２０２１抑制ＰＡＦ的刺激作用，抑制作用也呈剂量依赖性，５×１０￣（－５）Ｍ／ＬＢＮ５２０２１完全抑制ＧＭＣ合成活性氧。本研究说明ＰＡＦ可诱导ＧＭＣ合成活性氧。     

血小板T细胞活化抗原1分子在NK细胞上的作用研究

目的 研究血小板T细胞活化抗原1（PTA1）分子在NK细胞杀伤过程中的作用。方法 应用重民地向细胞毒实验,探讨了PTA1分子对混合淋巴细胞反应中活化的NK细胞杀伤作用的影响。结果 在重导向细胞毒实验中,PTA1 McAb能明显上调NK细胞及CTL的杀伤活性。结论 PTA1分子在NK细胞上具有刺激性受体的功能。     

Cyclosporin A inhibits T cell receptor-induced interleukin-2 synthesis of human T lymphocytes by selectively preventing a transmembrane signal transduction pathway leading to sustained activation of a protein kinase C isoenzyme,protein kinase C-β

Stimulation of human peripheral blood lymphocytes via T cell receptor/CD3 complex resulted in a bimodal activation of protein kinase(s) C (PKC). Within 10 min of stimulation PKC-α was translocated to, and thus activated in, the plasma membranes of human lymphocytes, followed by a fast dissociation of this isotype from the plasma membrane. This short term activation and translocation PKC-α proved to be cyclosporin A (CsA) insensitive. After 90 min of stimulation PKC-β was translocated to and remained bound to the plasma membranes for up to 4 h. Preincubation of human lymphocytes with 200 ng/ml CsA specifically and completely abolished the sustained activation of PKC-β. Neither the phorbol ester-induced direct activation of PKC nor the specific activity of the plasma membrane-bound enzyme was influenced by CsA, suggesting that a signal transduction pathway leading to sustained activation of PKC-β was influenced by the immunosuppressive agent. In fact, CsA inhibited, in a concentration-dependent manner, the activation of lysophosphatid acyltransferase-catalyzed elevated incorporation of cis-polyunsaturated fatty acids into plasma membrane phospholipids. While interleukin-2 (IL-2) synthesis and cellular proliferation were completely inhibited by 200 ng/ml CsA in BMA 030- or BMA 031-stimulated cells, expression of high-affinity IL-2 receptors was not influenced by the immunosuppressive drug. These results suggest that synthesis and expression of high-affinity IL-2 receptors might be regulated by a signal-transducing pathway involving activation and translocation of PKC-α. Lysophosphatid acyltransferase-catalyzed incorporation of cis-polyunsaturated fatty acids might represent another mechanism of signal transduction implicated in the activation and translocation of PKC-β, which is specifically inhibited by CsA. Neutralization of PKC-β by introducing anti-PKC-β antibodies prevented IL-2 synthesis and proliferation in stimulated human lymphocytes. The results suggest a possible link between activation of PKC-β and regulation of IL-2 synthesis in activated human lymphocytes. Thus, inhibition of the activation and translocation of PKC-β by CsA may result in inhibition of IL-2 gene expression in human lymphocytes.     

Protein kinase C in IL- 2 signal transduction

Interleukin-2 (IL-2), secreted principally by activated helper T-cells, plays a pivotal role in the generation and regulation of the immune response. The various biologic functions of IL-2 have been the focus of intensive study over the years and have been well worked out. By contrast, an understanding of the intracellular signals coupled to the IL-2 receptor and responsible for mediating IL-2 effects in T-cells is far less developed, and the role that protein kinase C (PKC) may play in the various cellular responses to IL-2 receptor activation is unclear. In this article we will discuss IL-2, its receptors, and IL-2 signal transduction in relation to the physiological roles PKC activation may play in IL-2-mediated activation of T-cells and other hematopoietic cells.     

人甲状腺癌组织中蛋白激酶C和蛋白激酶A活性变化的实验研究

本研究测定了8例恶性甲状腙癌病人和7例良性甲状腺瘤病人手术切除的肿瘤组织中蛋白激酶C（PKC)和蛋白激酶A（PKA）的活性变化，结果显示：恶性甲状腺癌组织中胞膜和胞浆PKC括性分剐比良性甲状腺瘤组织中胞膜和胞浆PKC恬性高14倍和23倍；恶性性甲状腺癌组织中PKA活性比良性甲状腺瘤组织中PKA恬性高2.6倍：恶性性甲状腺癌组织中PKC：PKA比值为4.2良性甲状腺瘤组织中PKC：PKA比值为4.2良性甲状腺瘤组织中PKC：PKA比值为0.53。PKC:PKA比值为0．53．PKC和PKA的激活是甲状腺癌细胞恶性增殖的原因之一，PKC和PKA都参予甲状腺癌细胞增殖过程的细胞内信息传递．而其中PKC的作用似乎更为重要。     

Differential activation of T cell cytokine production by the extracellular signal-regulated kinase (ERK) signaling pathway

Stimulation of T cells via the T cell receptor (TCR) activates a number of signaling pathways that are potentially involved in the elicitation of physiological responses, such as the production of cytokines. The extracellular signal-regulated kinases (ERK) are a group of molecules activated in response to TCR ligation, whose role in T cell cytokine production is controversial. In this study, we have asked whether ERK activation is coupled to the production of a number of T cell-derived cytokines, and whether particular cytokines are differentially affected by ERK activation. To address these questions, we have utilized a constitutively active version of the immediate upstream activator of both ERK1 and ERK2, mitogen-activated/extracellular signal-regulated kinase 1 (MEK1), to activate ERK signaling selectively in the absence of other TCR-activated signaling pathways. The effect of constitutive MEK/ERK activation on T cell cytokine production was measured by transiently co-transfecting newly activated mouse T cells with DNA encoding constitutively active MEK1 (CA-MEK1) and the human interleukin-2 (IL-2) receptor α chain (hCD25), purifying hCD25⁺ transfectants by flow-cytometric cell sorting, and measuring the production of IL-3, IL-4, interferon (IFN)-γ and granulocyte/macrophage-colony-stimulating factor (GM-CSF) either in the presence or absence of ionomycin stimulation. Newly activated T cells were used in these experiments as they more closely resemble T cells activated in vivo than do transformed T cells or long-term established T cell clones. CA-MEK1 expression led to constitutive ERK activation, which acted synergystically with ionomycin treatment to stimulate cytokine production. Furthermore, these experiments revealed a hierarchy of cytokine responsiveness to MEK/ERK activation, such that the production of IL-3 was most affected, followed by GM-CSF, IFN-γ, and IL-4.     

Cyclic AMP-dependent protein kinase (cAK) in human B cells: co-localization of type I cAK (RIα2C2) with the antigen receptor during anti-immunoglobulin-induced B cell activation

Cyclic AMP (cAMP) inhibits antigen-stimulated B cell proliferation through activation of cAMP-dependent protein kinases (cAK). We have examined the molecular composition and cellular localization of cAK in human B cells. We find that human B cells contain substantial amounts of mRNA for RIα, RIIα, Cα and Cβ, barely detectable levels of RIβ mRNA, and no detectable RIIβ or Cγ mRNA. At the protein level, using Western blotting and subunit-specific antibodies against the different R subunits, we find RIα and RIIα, but no RIβ or RIIβ. The presence of catalytic subunits was demonstrated using a nonselective anti-C antiserum. By photoaffinity labeling of R subunits with 8-azido-[³²P]cAMP, followed by immunoprecipitation with subunit-specific antibodies, we were also able to demonstrate low levels of RIβ. Immunofluorescence staining of RIα and RIIα demonstrates a rather homogeneous intracellular (but extranuclear) distribution of RIα, whereas the RIIα subunits of cAK are localized to distinct perinuclear structures, previously identified as centrosomes in other cell types. Upon anti-Ig-mediated capping of B cells, RIα subunits redistribute to the cap, co-localizing with the antigen-receptors, whereas the intracellular localization of RIIα subunits remains unchanged.     

Involvement of protein kinase C in the galanin-induced antinociception in the brain of rats

Previous study in our laboratory demonstrates that microinjection of galanin into the arcuate nucleus of hypothalamus produced antinociceptive effects in rats. In the present study we investigated the involvement of protein kinase C (PKC) and PKC signaling pathways in the galanin-induced antinociception in the brain of rats. Intracerebroventricular injection of galanin produced antinociceptive effects in rats tested by hot-plate and Randall Selitto test. Interestingly, the galanin-induced antinociception was significantly attenuated by intracerebroventricular injection of the PKC inhibitor chelerythrine, indicating an involvement of PKC in the galanin-induced antinociception in rats. Taken together, the results demonstrate that galanin induces antinociceptive effects in the rat brain, and PKC is involved in the galanin-induced antinociception in the brain of rats.     

Effect of activation of protein kinase C on excitation-contraction coupling in frog twitch muscle fibres

Intracellular Ca²⁺ transients were recorded from frog twitch muscle fibres in response to voltage-clamp depolarizing pulses, using arsenazo III as an intracellular Ca²⁺ indicator. The effect of the activation of protein kinase C (PKC) on the Ca²⁺ transients was studied. With 1 M phorbol 12,13-dibutyrate (PDBu), a PKC activator, the peak of the Ca²⁺ transients increased to about 120% of control during the first 0.5 h, and then decreased gradually to a plateau of 44% of control within the following 2 h. This effect of PDBu could be alleviated significantly by PKC inhibitors, 10 M polymyxin B (PMB) or 30 M 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7). Moreover, PDBu caused an upward shift of the strength/duration curve. In Li⁺-loaded muscle fibres the Ca²⁺ transients could not fully recover after 80 mM K⁺ exposure for 15 min, while the post-K⁺ Ca²⁺ transients could be completely restored in the fibres not loaded with Li⁺. In the presence of 10 M PMB or 30 M H-7, a full restoration of the post-K⁺ Ca²⁺ transients was seen in Li⁺-loaded fibres. PMB supplemented after high-K⁺ exposure also could result in a complete recovery of the post-K⁺ Ca²⁺ transients in Li⁺-loaded fibres. The role of PKC in modulating excitation-contraction coupling in frog twitch muscle fibres is clearly indicated, but the mechanism(s) and physiological significance remain to be established.     

Selective modulation of specific protein kinase C (PKC) isoforms in primary human megakaryocytic vs. erythroid cells

We have investigated the pattern of expression of classical (α, βI, βII, γ), novel (δ) and atypical (ζ) protein kinase C (PKC) isoforms during the course of human hematopoietic differentiation along the closely related megakaryocytic and erythroid lineages. Using in situ immunofluorescence analysis, freshly isolated human pluripotent CD34⁺ hematopoietic progenitor cells expressed detectable amounts of all the PKC isoforms investigated. On the other hand, clear‐cut differences in terms of PKC staining were noticed between cells belonging to the erythroid and megakaryocytic lineages, obtained after 9 days of serum‐free liquid culture in the presence of specific growth factors. Specifically, 1) erythroid cells showed a very weak expression of PKC‐α, ‐βI, ‐βII, and ‐γ, while megakaryocytes showed an enhanced expression of all classical PKC isoforms, predominantly confined to the cytoplasm; 2) the expression of PKC‐δ increased in the cytoplasmic and nuclear compartments of both erythroid and megakaryocytic cells with respect to CD34⁺ cells; and 3) atypical PKC‐ζ isoform showed a striking accumulation in the nucleus during both erythroid and megakaryocytic differentiation. Anat Rec 255:7–14, 1999. © 1999 Wiley‐Liss, Inc.     

Lectin-induced increase in microvascular permeability to colloidal carbonin vitro may involve protein kinase C activation

Two plant lectins, wheat germ agglutinin (WGA) and concanavalin A (Con A), which are known to bind to endothelial cells (ECs), were found to increase the leakage of colloidal carbon (CC) into the walls of microvessels in the villi of rat small intestine, when added to a gelatin-containing perfusate (GPSS) at a concentration of 10 g/ml. Pretreatment of the microvessels with the protein kinase C (PKC) inhibitor Ro 31-8220 (1×10^–6 M) significantly reduced this effect. In contrast, the leakage of CC in response to A23187 (1×10^–4 M) was not affected by Ro 31-8220. Peanut agglutinin (PNA) and succinyl concanavalin A (SuccCon A), which do not bind to ECs, had no effect at a concentration of 10 g/ml. A lower concentration of WGA (1 g/ml) had no significant effect of its own, but significantly reduced the leakage of CC in response to both platelet-activating factor (PAF, 5×10^–6 M) and 5-hydroxytryptamine (5-HT, 1×10^–4 M), but not to -phorbol 12,13-dibutyrate (PDB, 1×10^–6 M). These results suggest that all these effects of WGA and Con A involve cell surface receptors, albeit in a nonspecific way. A possible mode of action is discussed.     

The role of protein kinase C in the regulation of extracellular signal-regulated kinase by the T cell antigen receptor

The aim of this study was to explore the role of protein kinase C (PKC) in the activation of mitogen-activated protein kinases (MAPK) in T lymphocytes. The MAPK extracellular signal-regulated kinase-2 (ERK2) is activated in response to phorbol esters which stimulate PKC, by transient expression of a constitutively active ras mutant, by cell activation via the G protein-coupled type 1 muscarinic acetylcholine receptor (HM1R) or in response to triggering of the T cell antigen receptor (TCR). The relative contribution of PKC to TCR and HM1R regulation of ERK2 was explored by examining the effects of a PKC inhibitor (Ro 31-8425) on ERK2 activation. The data demonstrate that phorbol ester and HM1R regulation of ERK2 was prevented by the PKC inhibitor, but that the inhibitor had no effect on ERK2 activation induced by expression of a constitutively active ras mutant p21v-Ha-ras. Furthermore, the TCR stimulates both PKC and p21^ras but TCR regulation of ERK2 was only weakly suppressed by the PKC inhibitor. These data indicate that PKC has a potential but not a predominant role in TCR regulation of ERK2.