Carrier-mediated uptake of the endogenous cannabinoid anandamide in RBL-2H3 cells

Anandamide (N-arachidonylethanolamide) is an endogenous cannabinoid that mimics the pharmacologic effects of Delta(9)-tetrahydrocannabinol, the major bioactive substance in marijuana. Anandamide appears to be synthesized, released, and inactivated by mechanisms similar to those for other neurotransmitters. Of interest to the present studies are reports that anandamide undergoes carrier-mediated uptake into neuronal or glial cells after release, followed by rapid intracellular degradation by the intracellular fatty acid amidohydrolase. In addition to effects in the brain, anandamide has multiple effects in the periphery, particularly on cells of the immune system that express both a peripheral cannabinoid receptor and amidohydrolase enzyme. We have performed a detailed characterization of anandamide uptake in the cognate mast cell line RBL-2H3 to test the hypothesis that the uptake system in peripheral cells is also carrier-mediated and functionally similar to that observed in the central nervous system. RBL-2H3 cells exhibited robust, saturable transport of [(3)H]anandamide that was both time- and temperature-sensitive. This transport activity was not dependent on extracellular ion gradients for uptake and was inhibited selectively by other fatty acid-derived molecules, anandamide congeners, and the psychoactive cannabinoids such as Delta(9)-tetrahydrocannabinol. We conclude that anandamide transport in the RBL-2H3 cells is carrier-mediated, and uptake in peripheral cells is functionally and pharmacologically identical with that observed in neurons and astrocytes.     

Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells

Aggregation of the high affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells results in rapid tyrosine phosphorylation and activation of Syk, a cytoplasmic protein tyrosine kinase. To examine the role of Syk in the Fc epsilon RI signaling pathway, we identified a variant of RBL-2H3 cells that has no detectable Syk by immunoblotting and by in vitro kinase reactions. In these Syk-deficient TB1A2 cells, aggregation of Fc epsilon RI induced no histamine release and no detectable increase in total cellular protein tyrosine phosphorylation. However, stimulation of these cells with the calcium ionophore did induce degranulation. Fc epsilon RI aggregation induced tyrosine phosphorylation of the beta and gamma subunits of the receptor, but no increase in the tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2 and no detectable increase in intracellular free Ca2+ concentration. By transfection, cloned lines were established with stable expression of Syk. In these reconstituted cells, Fc epsilon RI aggregation induced tyrosine phosphorylation of phospholipase C- gamma 1 and phospholipase C-gamma 2, an increase in intracellular free Ca2+ and histamine release. These results demonstrate that Syk plays a critical role in the early Fc epsilon RI-mediated signaling events. It further demonstrates that Syk activation occurs downstream of receptor phosphorylation, but upstream of most of the Fc epsilon RI-mediated protein tyrosine phosphorylations.     

Luteolin suppresses 5-hydroxytryptamine elevation in stimulated RBL-2H3 cells and experimental colitis mice

Increased 5-hydroxytryptamine may be associated with the development and progression of inflammatory bowel disease. In this study, we examined the suppressive effect of flavonoids on the increased intra- and extracellular 5-hydroxytryptamine levels in rat mast RBL-2H3 cells, known to produce 5-hydroxytryptamine by the phorbol 12-myristate 13-acetate stimulation. Among the flavonoids examined, luteolin and quercetin significantly reduced the cellular 5-hydroxytryptamine concentration. Gene and protein expression analyses revealed that luteolin significantly suppressed cellular tryptophan hydroxylase 1 expression induced by phorbol 12-myristate 13-acetate stimulation. Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling was also suppressed by luteolin, suggesting that this pathway is one of targets of 5-hydroxytryptamine modulation by luteolin. An in vivo experimental colitis model was prepared by administering 2.5% dextran sodium sulfate in drinking water to C57BL/6 mice for seven days. The ingestion of 0.1% dietary luteolin suppressed the increasing 5-hydroxytryptamine in the colorectal mucosa. In conclusion, luteolin possesses a suppressive effect on extensive 5-hydroxytryptamine formation in both experimental RBL-2H3 cells and colitis models.     

大鼠RBL-2H3细胞瞬时感受器电位M7通道参与细胞存活及凋亡

背景：瞬时感受器电位M7是肥大细胞上重要的钙离子通道,但其在肥大细胞存活及凋亡过程中的作用仍未明确。目的：观察瞬时感受器电位M7通道对大鼠RBL-2H3细胞存活及凋亡的影响。方法：取对数生长期RBL-2H3细胞,①分别以50,100,200μmol/L瞬时感受器电位通道阻断剂2-APB进行干预,并以0.1%DMSO干预的细胞及正常培养的细胞作对照。②以瞬时感受器电位M7-siRNA反转录病毒载体转染RBL-2H3细胞,并设立空载体组和正常对照组。结果与结论：经过100,200μmol/L的2-APB作用72h后,RBL-2H3细胞数减少,吸光度值降低（P〈0.05）,细胞凋亡增多,早期凋亡率和总凋亡率增加（P〈0.05）;RBL-2H3细胞转染si-瞬时感受器电位M772h后,吸光度值降低（P〈0.05）,细胞凋亡增多,早期凋亡率和总凋亡率增加（P〈0.05）。说明瞬时感受器电位M7通道参与了RBL-2H3细胞存活和凋亡过程。     

大鼠RBL-2H3细胞瞬时感受器电位M7通道参与细胞存活及凋亡

背景:瞬时感受器电位M7是肥大细胞上重要的钙离子通道,但其在肥大细胞存活及凋亡过程中的作用仍未明确。目的:观察瞬时感受器电位M7通道对大鼠RBL-2H3细胞存活及凋亡的影响。方法:取对数生长期RBL-2H3细胞,①分别以50,100,200μmol/L瞬时感受器电位通道阻断剂2-APB进行干预,并以0.1%DMSO干预的细胞及正常培养的细胞作对照。②以瞬时感受器电位M7-siRNA反转录病毒载体转染RBL-2H3细胞,并设立空载体组和正常对照组。结果与结论:经过100,200μmol/L的2-APB作用72h后,RBL-2H3细胞数减少,吸光度值降低(P<0.05),细胞凋亡增多,早期凋亡率和总凋亡率增加(P<0.05);RBL-2H3细胞转染si-瞬时感受器电位M772h后,吸光度值降低(P<0.05),细胞凋亡增多,早期凋亡率和总凋亡率增加(P<0.05)。说明瞬时感受器电位M7通道参与了RBL-2H3细胞存活和凋亡过程。     

糖皮质激素对于活化RBL-2H3表达IL-5的影响

目的:探讨糖皮质激素对于活化RBL-2H3(简称RBL)表达白细胞介素5(IL-5)的影响。方法:利用抗原和钙离子载体A23187分别激活RBL表达IL-5,通过总RNA提取、逆转录和半定量PCR研究加入糖皮质激素后对于RBL活化表达IL-5的影响。结果:经A23187活化的RBL,加入10-7mol/L地塞米松后,其IL-5表达量降低到对照的9%,加入10-6mol/L氢化可的松后,其IL-5表达量降低到对照的8.5%。经抗原活化的RBL,加入10-6mol/L地塞米松后,其IL-5表达量降低到对照的12%,加入10-6mol/L氢化可的松后,其IL-5表达量降低到对照的9%。结论:糖皮质激素对于活化RBL表达IL-5具有极大的抑制作用,提示糖皮质激素在临床上治疗哮喘的作用与其抑制肥大细胞表达IL-5存在一定关系。     

SynaptotagminⅡ在大鼠嗜碱性白血病细胞胞吐中的调节作用研究

目的 研究Synaptotaganin Ⅱ（简称Syt2）在RBL-2H3（简称RBL）胞吐中的作用。方法利用钙离子载体和抗原分别刺激Syt2＋和Syt2-（分别为Syt2过表达和表达减弱的RBL），诱导其活化而分泌，检测分泌的β-氨基已糖苷酶、组织蛋白酶D和5-羟色胺，分析Syt2对于细胞分泌的影响。结果 与对照相比，Syt2＋分泌的β-氨基已糖苷酶无明显区别，而Syt2-分泌的β-氨基已糖苷酶增加达3．5倍，结果说明Syt2的过表达对β-氨基已糖苷酶分泌无影响，而Syt2表达减弱却促进β-氨基已糖苷酶的分泌。与对照相比，Syt2＋分泌的组织蛋白酶D减少近一半，而Syt2-分泌的组织蛋白酶D增加近1倍，说明Syt2的表达量与组织蛋白酶D分泌量成负相关关系。与对照相比，Syt2＋分泌的5-羟色胺无明显差别，而Syt2-分泌的5-羟色胺有少量增加，说明Syt2的表达量对于5-羟色胺分泌有轻微影响。结论 Syt2主要对于RBL溶酶体胞吐起负调控作用。     

反转录病毒介导靶向瞬时感受器电位M7基因siRNA抑制RBL-2H3细胞的活化

背景:钙离子在肥大细胞活化后的脱颗粒反应起重要作用。瞬时感受器电位M7(transient receptor potential melastatin7,TRPM7)是肥大细胞重要的候选通道。目的:构建携带大鼠靶向TRPM7-siRNA的反转录病毒载体并检测其对大鼠RBL-2H3细胞抗原活化的影响。方法:实验设计3个TRPM7-siRNA序列和1个无关对照序列,克隆到酶切的pSuper-retro-neo-GFP反转录病毒载体,用重组质粒pSuper-retro-neo-GFP-shTRPM7-(1,2,3)采用脂质体Lipofectamine 2000转染RBL-2H3细胞,采用Western blot检测干扰效率。筛选出最有效的pSuper-retro-neo-GFP-siTRMP7与包装质粒共转染293FT细胞生成反转录病毒并感染RBL-2H3细胞,荧光实时定量PCR及Western blot检测TRPM7-siRNA的沉默效果。检测β-氨基已糖苷酶活性探讨RBL-2H3细胞抗原活化程度的改变。结果与结论:转染后的各组细胞中siTRPM7-3转染组的沉默效率最高(P<0.05)。pSuper-retro-neo-GFP-siTRMP7-3干扰组的TRPM7基因的mRNA水平和蛋白水平显著下调,致敏后其β-氨基已糖苷酶活性明显降低(P<0.05)。结果提示,降低TRPM7基因的表达可抑制RBL-2H3细胞的抗原活化。     

反转录病毒介导靶向瞬时感受器电位M7基因siRNA抑制RBL-2H3细胞的活化

背景：钙离子在肥大细胞活化后的脱颗粒反应起重要作用。瞬时感受器电位M7（transient receptor potential melastatin7,TRPM7）是肥大细胞重要的候选通道。目的：构建携带大鼠靶向TRPM7-siRNA的反转录病毒载体并检测其对大鼠RBL-2H3细胞抗原活化的影响。方法：实验设计3个TRPM7-siRNA序列和1个无关对照序列,克隆到酶切的pSuper-retro-neo-GFP反转录病毒载体,用重组质粒pSuper-retro-neo-GFP-shTRPM7-（1,2,3）采用脂质体Lipofectamine 2000转染RBL-2H3细胞,采用Western blot检测干扰效率。筛选出最有效的pSuper-retro-neo-GFP-siTRMP7与包装质粒共转染293FT细胞生成反转录病毒并感染RBL-2H3细胞,荧光实时定量PCR及Western blot检测TRPM7-siRNA的沉默效果。检测β-氨基已糖苷酶活性探讨RBL-2H3细胞抗原活化程度的改变。结果与结论：转染后的各组细胞中siTRPM7-3转染组的沉默效率最高（P〈0.05）。pSuper-retro-neo-GFP-siTRMP7-3干扰组的TRPM7基因的mRNA水平和蛋白水平显著下调,致敏后其β-氨基已糖苷酶活性明显降低（P〈0.05）。结果提示,降低TRPM7基因的表达可抑制RBL-2H3细胞的抗原活化。     

As2O3对K562细胞BCR/ABL蛋白酪氨酸磷酸化的影响

目的　进一步阐明Ａｓ２Ｏ３ 诱导Ｋ５６２ 细胞凋亡和抑制其生长的可能机制,为Ａｓ２Ｏ３ 在临床上的应用提供理论依据。方法　采用免疫沉淀、Ｗｅｓｔｅｒｎ ｂｌｏｔ、生物化学及免疫荧光等方法研究了Ａｓ２Ｏ３对ＢＣＲ／ＡＢＬ蛋白酪氨酸磷酸化及其所介导的信号途径和某些凋亡相关蛋白表达的影响。结果　１μｍｏｌ／ＬＡｓ２Ｏ３ 使细胞内多种蛋白酪氨酸磷酸化减少,而且ＢＣＲ／ＡＢＬ蛋白自身酪氨酸磷酸化亦减少,但０ ．１ μｍｏｌ／ＬＡｓ２Ｏ３ 对蛋白酪氨酸磷酸化的影响不明显;Ａｓ２Ｏ３ 对蛋白酪氨酸磷酸酶（ＰＴＰ） 活性未见明显影响;Ａｓ２Ｏ３ 下调ＪＡＫ２ 蛋白的表达,但对ＳＴＡＴ１ 和ＳＴＡＴ２ 蛋白的表达以及ＳＴＡＴ１ 蛋白酪氨酸磷酸化无影响;Ａｓ２Ｏ３ 亦不影响凋亡相关蛋白Ｂｃｌ２、ＢｃｌｘＬ／Ｓ、Ｂａｘ、ＩＣＨ１Ｌ、ｐ５３、ＰＡＲＰ的表达,Ａｓ２Ｏ３ 亦使Ｋ５６２ 细胞的ＰＭＬ蛋白降解。结论　Ａｓ２Ｏ３ 可能通过减少细胞内某些蛋白,尤其是ＢＣＲ／ＡＢＬ蛋白酪氨酸磷酸化和（ 或）下调ＪＡＫ２ 蛋白的表达而干扰ＢＣＲ／ＡＢＬ致癌信号的传导,引起Ｋ５６２ 细胞凋亡和抑制其生长。     

IL-5 receptor-mediated tyrosine phosphorylation of SH2/SH3-containing proteins and activation of Bruton's tyrosine and Janus 2 kinases

Interleukin 5 (IL-5) induces proliferation and differentiation of B cells and eosinophils by interacting with its receptor (IL-5R) which consists of two distinct polypeptide chains, alpha and beta (beta c). Although both IL-5R alpha and beta c lack a kinase catalytic domain, IL- 5 is capable of inducing tyrosine phosphorylation of cellular proteins. We investigated the role of IL-5R alpha in tyrosine phosphorylation of molecules involved in IL-5 signal transduction, using an IL-5-dependent early B cell line, Y16 and transfectants expressing intact or mutant IL- 5R alpha together with intact beta c. The results revealed that the transfectants expressing truncated IL-5R alpha, which entirely lacks a cytoplasmic domain, together with beta c, showed neither protein- tyrosine phosphorylation nor proliferation in response to IL-5. This confirms that IL-5R alpha plays a critical role in protein-tyrosine phosphorylation which triggers cell growth. IL-5 stimulation results in rapid tyrosine phosphorylation of beta c and proteins containing Src homology 2 (SH2) and/or SH3 domains such as phosphatidyl-inositol-3 kinase, Shc, Vav, and HS1, suggesting their involvement in IL-5- mediated signal transduction. IL-5 stimulation significantly enhanced activities of Janus 2 and B cell-specific Bruton's tyrosine kinases (JAK2 and Btk) and increased the tyrosine phosphorylation of JAK2 kinase. These results and recent data on signaling of growth factors taken together, multiple biochemical pathways driven by tyrosine kinases such as JAK2 and Btk are involved in IL-5 signal transduction.     

人IL-5诱导的STAT3酪氨酸磷酸化

目的分析人早幼粒细胞白血病细胞内STAT3的酪氨酸磷酸化活化情况。方法培养人早幼粒细胞白血病细胞株HL-60,分别用浓度为0,1.0,10,100 ng/ml的人白细胞介素（hIL）-5刺激,然后利用特异性抗体,用免疫沉淀法、聚丙烯酰胺凝胶（SDS PAGE）电泳及Western Blot方法进行检测。结果检测到HL-60细胞内不同浓度的STAT3表达。结论一定浓度的人IL-5能同时诱导HL-60细胞内STAT3α和STAT3α的酪氨酸（Y705）磷酸化,且在一定范围内这种诱导作用与IL-5的浓度呈正相关。     

TA-270 [4-hydroxy-1-methyl-3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone], an anti-asthmatic agent,inhibits leukotriene production induced by IgE receptor stimulation in RBL-2H3 cells

A novel quinolinone derivative, TA-270 [4-hydroxy-1-methyl-3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone], has been shown to inhibit antigen-induced asthmatic responses including the early-phase bronchoconstriction in actively sensitized guinea pigs. Here we characterized the action mechanisms of TA-270 in cellular level in vitro. In RBL-2H3 mast cells sensitized with dinitrophenol (DNP)-specific IgE, the antigen exhibited several mast cell functions, including hexosaminidase release as a marker of degranulation, production of tumor necrosis factor-alpha, and production of immunologically detective leukotrienes. These antigen-induced actions were associated with the activation of several early signaling events, including inositol phosphate production reflecting phospholipase C activation and extracellular signal-regulated kinase activation. When the cells were treated with TA-270, the antigen-induced leukotriene production was almost completely suppressed, but other antigen-induced actions listed above were hardly affected. This drug also failed to affect the antigen-induced phospholipase A2 activation as evaluated by the total release of arachidonic acid and its metabolites from the cells prelabeled with radioactive arachidonic acid. However, TA-270 clearly changed the arachidonic acid metabolic pathway. It suppressed the accumulation of 5-lipoxygenase products, including leukotrienes, but hardly affected the accumulation of cyclooxygenase products. The inhibitory action of TA-270 on leukotriene production was also observed in human neutrophils and eosinophils. We conclude that TA-270 inhibits 5-lipoxygenase activity and, thereby, suppresses the antigen-induced leukotriene production.     

Ligation of VLA-4 on T cells stimulates tyrosine phosphorylation of a 105-kD protein

The VLA/integrins are a family of heterodimeric adhesion receptors shown to be involved in cell-to-cell and cell-to-extracellular matrix (ECM) interactions. Given recent evidence that VLA molecules can synergize with the CD3/T cell receptor (TCR) pathway to activate T cells, it is important to identify biochemical event(s) generated by these molecules. Here, we report that the engagement of VLA-4 on T cells with specific antibodies or its ligand activates protein-tyrosine kinase (PTK) activity as detected by antiphosphotyrosine immunoblotting. The crosslinking of VLA-beta 1 (CD29) with a specific monoclonal antibody (mAb) (anti-4B4) plus anti-mouse immunoglobulin resulted in the rapid tyrosine phosphorylation of a 105-kD protein (pp105) in the human T cell line H9, as well as in peripheral resting T cells. The increase in tyrosine phosphorylation of pp105 was specifically mediated by VLA-4, since mAbs against alpha 4, but not against other VLA alpha chains, could induce this phosphorylation. In addition, the binding of T cells with the CS1 alternatively spliced segment of fibronectin (the binding site recognized by VLA-4) induced pp105 tyrosine phosphorylation. Crosslinking the CD3 complex or VLA-4 molecules with mAbs demonstrated that each of these molecules stimulated the tyrosine phosphorylation of unique sets of proteins with different kinetics, suggesting that these two receptor systems are coupled to distinct PTKs. Since tyrosine phosphorylation of cellular proteins has been shown to be a crucial biochemical event in cell growth, our findings suggest that the induction of pp105 tyrosine phosphorylation via VLA-4 may play a role in the transduction of activation signals through this molecule.     

Methylxanthines block antigen-induced responses in RBL-2H3 cells independently of adenosine receptors or cyclic AMP: evidence for inhibition of antigen binding to IgE.

Activation of a novel adenosine receptor in a rat tumor mast cell line (RBL-2H3 cells) elicits a transient generation of inositol 1,4,5-trisphosphate and an equally transient increase in the level of free cytosol Ca++: Such responses promote little exocytosis, but markedly enhance the secretory response to antigen. A variety of xanthine adenosine receptor antagonists did not suppress the responses to the adenosine analog 5-N-ethylcarboxamidoadenosine. However, 3-isobutyl-1-methylxanthine (IBMX) and certain related xanthines inhibited antigen (dinitrophenylated bovine serum albumin, DNP-BSA)-induced generation of inositol phosphates, the increase in level of free cytosolic Ca++ and exocytosis in RBL-2H3 cells that were primed with a monoclonal DNP-specific immunoglobulin E (from hybridoma H1 DNP-epsilon-26.82). The same compounds inhibited the binding of antigen to cell attached DNP-specific IgE in a highly selective manner. Incorporation of an aromatic or cycloalkyl group in the 8-position of IBMX or theophylline, for example, resulted in compounds that were more potent inhibitors than the parent compounds. Conversely, substituents in the 7- or 9-position of IBMX resulted in inactive compounds. 1,3-Diethylxanthine and 1,3-dipropylxanthine had no activity, suggesting that substituents as large as ethyl or propyl are not tolerated at the 1-position. Inhibition by IBMX was not observed when cells were activated by nonimmunological stimulants or when cells were primed with certain other monoclonal preparations of DNP-specific IgE and stimulated by DNP-BSA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of phospholipase C in human B cells is dependent on tyrosine phosphorylation.

Cross-linking of the surface antigen receptor on B lymphocytes has been demonstrated to lead to activation of phospholipase C (PLC) with subsequent increases in production of inositol phosphates and diacylglycerol. In turn, these second messengers increase cytosolic free calcium [( Ca2+]i) and activate the serine threonine phosphotransferase protein kinase C (PKC). These processes are thought to play a major role in B cell activation and proliferation. However, the mechanism linking the B lymphocyte antigen receptor to phospholipase C remains to be identified. We demonstrate herein that activation of the antigen receptor on human lymphocytes, in addition to activation of PLC, increases tyrosine phosphorylation of specific substrates. Tyrphostins, a new class of tyrosine kinase inhibitors which compete for substrate binding site of specific tyrosine kinases have recently been synthesized. Preincubation of B lymphocytes with two different tyrphostins blocked anti-IgM-induced proliferation, oncogene expression, tyrosine phosphorylation, increases in [Ca2+]i, and production of inositol phosphates. The same inhibitors were without effect on B cell proliferation induced by phorbol esters and cation ionophores which directly activate PKC and increase [Ca2+]i thus bypassing PLC. These findings strongly indicate that tyrphostins do not exhibit significant nonspecific toxicity and suggest that they act proximal to PLC. The ability of the tyrphostins to block increases in [Ca2+]i and inositol phosphate production, after activation of the B cell antigen receptor, indicates that a tyrosine kinase acts as an essential link between the B cell antigen receptor and PLC.     

H1-A extracted from Cordyceps sinensis suppresses the proliferation of human mesangial cells and promotes apoptosis,probably by inhibiting the tyrosine phosphorylation of Bcl-2 and Bcl-XL

H1-A, a pure compound used in traditional Chinese medicine, is effective in the treatment of autoimmune disorders of MRL lpr/lpr mice. We have previously reported that after 8 weeks of oral therapy with H1-A, 40 microg/kg/day, MRL lpr/lpr mice demonstrated significantly less proteinuria, lower serum creatinine levels, and less renal mesangial proliferation than mice in an untreated group. To clarify the pharmacologic properties of H1-A, we studied its cellular and subcellular effects in cultured human mesangial cells. Our results show that H1-A inhibits cell proliferation and promotes the apoptosis of interleukin (IL)-1- and platelet-derived growth factor (PDGF)-BB-activated human mesangial cells in vitro. Uptake of tritiated thymidine was nearly totally suppressed by the addition of 12.5 micromol/L H1-A (counts per minute decreased from 3905 +/- 70 to 141 +/- 5). The population of S-phase cells decreased from 15.5% +/- 1.7% to 10.0% +/- 0.3%, and G0 + G1 phase cells increased from 68.8% +/- 0.07% to 74.6% +/- 0.05%. This suppression was not a result of cytotoxicity. Apoptosis of human mesangial cells was detectable after treatment with 12.5 or 25 micromol/L H1-A. Using immunoprecipitation and immunoblotting, we found that H1-A inhibits tyrosine phosphorylation of human mesangial proteins and that Bcl-2 and Bcl-XL were probably among these proteins. These findings suggest that H1-A modulates some subcellular signal-transduction pathways and changes the balance between proliferation and apoptosis of mesangial cells in vitro or in vivo. H1-A may be effective in the management of autoimmune disorders, and the modulation of the signal transduction proteins Bcl-2 and Bcl-XL may represent a target for future pharmacologic interventions.     

粒—巨噬系集落刺激因子和红细胞生成素诱导JAK2酪氨酸磷酸化…

目的：确定与细胞内蛋白质酪氨酸磷酸化相关的激酶。方法：用粒－巨噬系集落刺激因子（ＧＭ－ＣＳＦ）和红细胞生纱（Ｅｐｏ）依赖细胞株ＵＴ－７以及Ｅｐｏ依赖细胞株ＵＴ－７／Ｅｐｏ进行酪氨酸磷酸化的研究。结果：当ＧＭ－ＣＳＦ或Ｅｐｏ刺激其生长依赖细胞时可快速诱导分子量为１４５０００，１３００００，８００００和４００００等多种蛋白质酷氨酸磷酸化。鉴定了分子量为１３００００的酪氨酸磷酸化的蛋白质为ＪＡＫ２，一种     

葡萄糖-6-磷酸脱氢酶缺乏时红细胞膜带3蛋白酪氨酸磷酸化的改变

目的：从红细胞膜蛋白磷酸化改变的角度探讨葡萄糖－6－磷酸脱氢酶（G6PD）缺乏症溶血的机制。方法：Western blot法检测G6PD缺乏症的红细胞膜蛋白磷酸化的改变以及二硫苏糖醇（DTT）对蛋白磷酸化的影响；以对硝基苯磷酸（PNPP）为底物，测磷酸酪氨酸磷酸酶（PTPs)活性以探讨磷酸化改变的可能成因。结果：G6PD缺乏的红细胞膜带3（Band 3)蛋白酪氨酸的磷酸化水平较正常对照明显增多，而PTPs活性检测较正常对照组明显减弱；DTT处理的G6PD缺乏红细胞，其膜Band 3蛋白酪氨酸的磷酸化与未处理者无明显差异，PTPs活性检测结果与未处理者亦无显著差异。结论：氧化致使G6PD缺乏红细胞的PTPs活性减弱，膜Band 3蛋白酪氨酸磷酸化增强，成为红细胞溶血的一个重要原因。但PTPs巯基的改变并不是影响PTPs活性的唯一因素。     

Analysis of tyrosine phosphorylation in resident peritoneal cells during diet restriction by laser scanning cytometry

Tyrosine phosphorylation plays a critical role in signal transduction pathways in immune cells. Laser scanning cytometer (LSC), a newly developed microscope-based cytofluorometer, may overcome shortcomings of Western blotting and flow cytometry in the detection of intracellular signaling transduction. The aims of this study were to visualize and quantitate intracellular phosphotyrosine in the peritoneal cells harvested from diet-restricted mice by LSC. In addition, using LSC, we identified the main cell type with activated tyrosine phosphorylation in response to an inflammatory stimulus and we investigated the intracellular distribution of tyrosine phosphorylation within the peritoneal macrophages. Mice were assigned to the ad libitum and diet-restricted, i.e., 75% restricted food intake, groups. After 7 days of pair feeding, the peritoneal cells were harvested. Tyrosine phosphorylation in the harvested cells with either N-formyl-methionyl-leucyle-phenylalanine (fMLP) or lipopolysaccharide (LPS) stimulation was examined using LSC. Tyrosine phosphorylation of peritoneal cells from the diet-restricted group was significantly higher than that from the ad libitum group, regardless of stimulation. Stimulation of peritoneal cells with either fMLP or LPS significantly increased tyrosine phosphorylation in the ad libitum group, but not in the diet-restricted group. The relocation feature of LSC revealed that the cells with distinct tyrosine phosphorylation were macrophages. Topographic analysis demonstrated that phosphotyrosine was localized mainly in the cytoplasm of these cells. In summary, LSC revealed that tyrosine phosphorylation is mainly in the cytoplasm of the peritoneal macrophages and is deranged by diet restriction. LSC is a powerful tool for the study of intracellular signaling transduction.