高原低氧习服大鼠红细胞变形性的变化规律及其分子机制

目的：探讨高原低氧习服大鼠红细胞变形性的变化规律及其分子机制。方法：将健康雄性大鼠随机分为3组（n=10）：常氧对照组、急性低氧组和低氧习服组。模拟高原低氧环境对大鼠分别进行急性低氧和间断低氧习服,麻醉后心脏采血,分别测定大鼠红细胞变形性、膜流动性、膜胆固醇和总磷脂含量、膜磷脂成分的含量、红细胞ATP酶活性、红细胞内Na＋和Ca2＋浓度及建立红细胞膜蛋白质双向电泳图谱,寻找差异蛋白质点,对其进行质谱鉴定。结果：①急性低氧大鼠红细胞变形性、膜流动性、膜胆固醇和总磷脂含量、红细胞ATP酶活性均降低;红细胞内Na＋和Ca2＋浓度均增高;红细胞膜磷脂酰丝氨酸（PS）、鞘磷脂（SM）含量增加,磷脂酰胆碱（PC）含量降低;建立了红细胞膜蛋白质双向电泳图谱,选取7个差异蛋白质点,其中4个在急性低氧后表达降低。②低氧习服大鼠红细胞变形性、膜流动性、膜胆固醇和总磷脂含量、红细胞ATP酶活性明显均增高;红细胞内Na＋和Ca2＋浓度均降低;红细胞膜PS、SM含量降低,PC含量增加;上述7个差异蛋白质点中4个在低氧习服后表达增高,3个表达降低,质谱技术鉴定结果为补体结合蛋白、水通道蛋白、膜攻击复合物抑制因子、葡萄糖运载体、脂质移行酶、氨基磷脂转移酶、依赖ATP的翻转酶,其中后三个酶与红细胞膜磷脂翻转有关。结论：急性低氧引起红细胞变形性、膜流动性、膜蛋白质表达、红细胞ATP酶活性及胞内Na＋和Ca2＋浓度方面相应的改变;经低氧习服后,上述指标有所改善,低氧习服对急性低氧引起红细胞的影响具有一定的保护作用;红细胞膜上的3种蛋白质,包括脂质移行酶、氨基磷脂转移酶和依赖ATP的翻转酶在低氧习服改善红细胞变形性的机制中可能发挥重要的作用。     

人红细胞生成素受体的研究进展

人红细胞生成素受体(hEPOR)是位于相对成熟阶段人体红系祖细胞表面的跨膜蛋白,它能专一性结合人红细胞生成素(hEPO),将促进细胞生长、增殖和分化的信号传导到膜内,该过程涉及了hEPOR自身及部分相关蛋白的磷酸化.hEPOR具有一些与其功能相关的保守结构,它的氨基酸序列与鼠EPOR(mEPOP)高度同源.EPOR因为膜外R129C突变或结合特定蛋白质而具有组成型活性.EPOR还与红白血病等多种血液病密切相关.     

pAkt在衰老细胞中的核转位

磷脂酰肌醇3 激酶（PI3K）/蛋白激酶B（PKB,又称为Akt）/叉头转录因子（FOXO）信号通路在从酵母菌到小鼠的寿命以及衰老的调节上都起着非常重要的作用．有研究发现,血清可以激活年轻细胞、衰老细胞胞浆内的Akt,并且年轻细胞核内磷酸化Akt(pAkt)增多,而衰老细胞核内pAkt没有增多．为了研究衰老细胞膜是否发生转位受损,即pAkt能否通过衰老细胞核膜进入核内,通过激光共聚焦显微镜（CLSM）、Western 印迹等实验方法,发现衰老细胞胞浆中pAkt可以进入核内,进入核内的pAkt很快被去磷酸化灭活．     

亚硒酸钠抗红细胞膜蛋白交联作用的机理探讨

邻苯二酚氧化处理人红细胞膜会导致膜蛋白交联,产生高分子聚合物(HMP)。用N—乙基马来酰胺(NEM)封闭膜蛋白硫基,则不产生HMP。预先用Na SeO_3(0.05mol/L)处理红细胞膜,也同样不产生HMP。用N—(3-芘)马来酰胺(N-〔3-P〕NEM)标记红细胞膜来测试不同浓度Na_2SeO_3对荧光强度的影响。结果表明,随着Na SeO_3浓度增高荧光强度相应降低。Na_2SeO_3对红细胞膜的预处理时间和荧光强度的变化有关。经Na SeO_3处理的红细胞膜ESR谱提示了Na_2SeO_3与材相互作用有关。用荧光法测定膜结合硒含量表明,Na_2SeO_3处理红细胞膜可导致膜结合硒含量增高。推测,Na SeO_3很可能与膜蛋白疏基作用形成结合硒,从而起到抗膜蛋白交联作用。     

严重烧伤早期红细胞膜胆固醇含量及Na+-K+-ATP酶活性的变化

目的 :研究严重烧伤患者早期红细胞滤过指数 (EFI)与红细胞膜胆固醇含量、Na K ATPase活性的变化。探讨其在严重烧伤早期中的相互关系及意义。方法 :采用核孔滤膜法测定红细胞滤过指数 (EFI) ,用化学修饰电极法测定红细胞膜胆固醇含量 ,应用定磷法测定红细胞膜Na K ATPase活性。结果 :4 7例严重烧伤早期患者EFI较 6 0例正常对照组下降 (P <0 .0 1) ,红细胞膜胆固醇含量、Na K ATPase活性均高于正常对照组 (P <0 .0 1) ,且红细胞膜胆固醇含量、Na K ATPase活性与EFI呈密切负相关 (rcho =- 0 .871,rATPase =- 0 .80 1,P <0 .0 1)。结论 :严重烧伤早期EFI下降 ,变形性明显减低是导致血液粘度和微循环改变的原因之一 ,红细胞膜胆固醇含量和Na K ATPase活性的变化则是引起EFI下降、变形性减低的重要因素     

竹红菌甲素对红细胞膜和几种磷脂脂质体膜的流动性的光敏损伤

本文以荧光探针为手段,通过测量膜偏振度的变化,探讨了竹红菌甲素光敏作用对红细胞膜和几种磷脂脂质体膜的流动性的损伤。结果表明,甲素光敏作用使不同种类的磷脂(DPPC,DPPC/DPPE,红细胞膜磷脂)脂质体的流动性增加,其对光敏作用的敏感程度为红细胞膜磷脂脂质体显著小于DPPC/DPPE脂质体及DPPC脂质体。对红细胞膜来说,甲素光敏作用使其流动性呈现先降低而后增加的现象。去除膜上的spectrin以及用胰蛋白酶处理可使这种流动性变化的幅度受到抑制。据此,我们认为,膜磷脂,膜蛋白对甲素光敏作用中膜流动性的变化有着不同的影响,膜蛋白,特别是spectrin,是其中极重要的因素。     

大强度训练及恢复后大鼠红细胞变形能力和红细胞膜蛋白的变化

目的 :探讨运动对红细胞变形性和红细胞膜蛋白的影响及其相互关系。方法 :设计不同强度的训练方案 ,用激光衍射法测定红细胞变形能力 ,用SDS PAGE方法测定一定体积大鼠红细胞膜中的重要蛋白带 3蛋白 (band 3)和肌动蛋白 (actin)的含量 ,研究运动即刻和恢复后红细胞变形性及膜蛋白的变化。结果 :长期的运动训练会促进大鼠红细胞变形能力的改善和红细胞膜band 3蛋白和actin的良好发展 ,一次大强度训练会引起红细胞膜band 3蛋白和actin含量的减少 ,大鼠红细胞变形能力降低 ,一周和二周的大强度训练会提高恢复期大鼠红细胞的变形能力和红细胞膜band 3蛋白和actin含量。结论 :运动训练造成的红细胞膜蛋白含量的变化 ,导致了红细胞膜结构的改变 ,从而影响红细胞变形能力 ,可能是训练对红细胞变形能力的作用机制之一。     

钒对人红细胞膜蛋白和膜脂质过氧化的影响

对钒酸根Ｖ（Ｖ）与红细胞膜相互作用研究表明Ｖ（Ｖ）使膜蛋白内源荧光淬火和膜巯基含量降低,但对膜脂质过氧化影响较小,提示Ｖ（Ｖ）主要与膜蛋白作用,与Ｖ（Ｖ）不同,Ｖ（Ⅳ）与红细胞膜的作用虽使膜蛋白巯基含量下降,但不显,其主要作用是引起膜脂质过氧化。     

羟自由基对人红细胞氧化囊泡化的作用

红细胞经氧化处理后,发现红细胞膜区带１、２、２．１及３易形成高聚物,同时Ｈｂ亦有氧化变性。氧化过程中磷脂（ＰＳ、ＰＥ）逐渐减少,根据此结果提出氧化产生囊泡化的机制是：首先Ｈｂ氧化变性,膜骨架蛋白聚集,从而网架松散,膜磷脂脱离骨架的束缚,膜脂质与膜整合蛋白形成小囊泡从红细胞上脱落。     

注射装载孕酮的红细胞膜泡诱发蟾蜍卵成熟的研究

中华大蟾蜍长足的卵母细胞,经注入装载水相孕酮的红细胞膜泡,能被诱发成熟;直接注入水相孕酮的卵母细胞,无能恢复成熟分裂。将蛋白酶处理的红细胞制备成装载水相孕酮的膜泡,注入卵内,照样能诱发其成熟分裂;然而,分别用根皮素结合或磷脂酶A_2水解红细胞膜磷脂,制备的膜泡,虽亦包裹着水相孕酮,但注射的卵母细胞都未能被诱发成熟。这些结果表明,在通过红细胞膜转运孕酮诱发卵母细胞成熟过程中,红细胞膜上的某些膜蛋白可能不是必要的成份,而膜磷脂类却是关键成份,它不仅可能保证孕酮不被迅速代谢,且保证孕酮从卵母细胞内部诱发成熟分裂。     

Mitosis-specific phosphorylation of vimentin by protein kinase C coupled with reorganization of intracellular membranes

Using two types of anti-phosphopeptide antibodies which specifically recognize vimentin phosphorylated by protein kinase C (PKC) at two distinct PKC sites, we found that PKC acted as a mitotic vimentin kinase. Temporal change of vimentin phosphorylation by PKC differed form changes by cdc2 kinase. The mitosis-specific vimentin phosphorylation by PKC was dramatically enhanced by treatment with a PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), while no phosphorylation of vimentin by PKC was observed in interphase cells treated with TPA. By contrast, the disruption of subcellular compartmentalization of interphase cells led to vimentin phosphorylation by PKC. Cytoplasmic and nuclear membranes are fragmented and dispersed in the cytoplasm and some bind to vimentin during mitosis. Thus, targeting of activated PKC, coupled with the reorganization of intracellular membranes which contain phospholipids essential for activation, leads to the mitosis-specific phosphorylation of vimentin. We propose that during mitosis, PKC may phosphorylate an additional subset of proteins not phosphorylated in interphase.     

A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C

Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of approximately 10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.     

Protamine induces autophosphorylation of protein kinase C: stimulation of protein kinase C-mediated protamine phosphorylation by histone.

Protein kinase C (PKC), a protein phosphorylating enzyme, is characterized by its need for an acidic phospholipid and for activators such as Ca2+ and diacylglycerol. The substrate commonly used in experiments with PKC is a basic protein, histone III-S, which needs the activators mentioned. However, protamine, a natural basic substrate for PKC, does not require the presence of cofactor/activator. We report here that protamine can induce the autophosphorylation of PKC in the absence of any PKC-cofactor or activator; this may represent a possible mechanism of cofactor-independent phosphorylation of this protein. It was investigated if protamine itself can act as a PKC-activator and stimulate histone phosphorylation in the manner of Ca2+ and phospholipids. Experiments however showed that protamine is not a general effector of PKC. On the contrary, histone stimulated PKC-mediated protamine phosphorylation and protamine-induced PKC-autophosphorylation. Histone alone did not induce PKC-autophosphorylation. Kinetic studies suggest that histone increases the maximal velocity (Vmax) of protamine kinase activity of PKC without affecting the affinity (Km). Other polycationic proteins such as polyarginine serine and polyarginine tyrosine were not found to influence PKC-mediated protamine phosphorylation, indicating that the observed effects are specific to histone, and are not general for all polycationic proteins. These results suggest that histone can modulate the protamine kinase activity of PKC by stimulating protamine-induced PKC-autophosphorylation.     

Protein kinase C delta (PKC delta): activation mechanisms and functions

Protein kinase C (PKC)delta was the first new/novel PKC isoform to be identified by the screening of mammalian cDNA libraries, based on the structural homology of its nucleotide sequences with those of classical/conventional PKC isoforms. PKC delta is expressed ubiquitously among cells and tissues. It is activated by diacylglycerol produced by receptor-mediated hydrolysis of membrane inositol phospholipids as well as by tumor-promoting phorbol ester through the binding of these compounds to the C1 region in its regulatory domain. It is also cleaved by caspase to generate a catalytically active fragment, and it is converted to an active form without proteolysis through the tyrosine phosphorylation reaction. Various lines of evidence indicate that PKC delta activated in distinct ways plays critical roles in cellular functions such as the control of growth, differentiation, and apoptosis. This article briefly summarizes the regulatory mechanisms of PKC delta activity and its functions in cell signaling.     

Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C

The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified. A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC. It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils. Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBD-phallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin. [32P]ATP was used to detect protein phosphorylation in electroporated cells. Exposure of neutrophils to 4 beta-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the actin cytoskeleton, whereas the 4a-isomer, which is inactive towards PKC, failed to produce any of these changes. Moreover, 1,2-dioctanoylglycerol, mezerein, and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, which are nonphorbol activators of PKC, also promoted actin assembly. Although these effects were consistent with a role of PKC, the following observations suggested that stimulation of conventional isoforms of the kinase were not directly responsible for actin assembly: (a) Okadaic acid, an inhibitor of phosphatases 1 and 2A, potentiated PMA-induced protein phosphorylation, but not actin assembly; and (b) PMA-induced actin assembly and membrane ruffling were not prevented by the conventional PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, staurosporine, calphostin C, or sphingosine at concentrations that precluded PMA-induced protein phosphorylation and superoxide production. On the other hand, PMA-induced actin assembly was inhibited by long-chain fatty acid coenzyme A esters, known inhibitors of nuclear PKC (nPKC). We conclude that PMA-induced actin assembly is unlikely to be mediated by the conventional isoforms of PKC, but may be mediated by novel isoforms of the kinase such as nPKC.     

Protein kinase C mediates platelet-derived growth factor-induced tyrosine phosphorylation of p42

One of the early events after stimulation of Swiss 3T3 cells with either platelet-derived growth factor (PDGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), diacylglycerol, or several other mitogens is the near stoichiometric phosphorylation at tyrosine and serine of a scarce cytoplasmic protein (p42). TPA and diacylglycerol are known to directly stimulate the activity of a protein-serine/threonine kinase, protein kinase C (PKC). PDGF and several other mitogens stimulate tyrosine kinases directly and PKC indirectly. We have therefore examined the involvement of PKC in p42 tyrosine phosphorylation in Swiss 3T3 cells. Firstly, six agents which stimulated phosphorylation of p42 also stimulated phosphorylation of a known PKC substrate, an 80,000-Mr protein (p80). Secondly, in PKC-deficient cells (cells in which PKC activity was reduced to undetectable levels by prolonged exposure to TPA), PDGF-induced p42 phosphorylation was reduced three- to fourfold. Phosphoamino acid analysis of phosphorylated p42 from PDGF-stimulated PKC-deficient cells revealed primarily phosphoserine and only a trace of phosphotyrosine, suggesting that the reduction in PDGF-stimulated tyrosine phosphorylation of p42 resulting from PKC deficiency is greater than three- to fourfold. Finally, comparison of antiphosphotyrosine immunoprecipitates of PKC-deficient versus naive cells revealed that most other PDGF-induced tyrosine phosphorylation events were quite similar. These data suggest that mitogens such as PDGF, which directly stimulate phosphorylation of some proteins at tyrosine, induce p42 tyrosine phosphorylation via a cascade of events involving PKC.     

Protein kinase C-mediated tyrosine phosphorylation of paxillin and focal adhesion kinase requires cytoskeletal integrity and is uncoupled to mitogen-activated protein kinase activation in human hepatoma cells

Treatment of cultured human hepatoma HepG2 cells with the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), results in an increase in tyrosine phosphorylation of several proteins, including the focal adhesion kinase (FAK) and paxillin using anti-phosphotyrosine Western blotting and immunoprecipitation. However, when cells are in suspension or in the presence of cytochalasin D which disrupts the intracellular network of actin microfilaments, TPA loses its ability to stimulate tyrosine phosphorylation of FAK and paxillin but it still activates mitogen-activated protein kinase (MAPK) and induces PKC translocation from cytosol to the membrane in HepG2 cells. On the other hand, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, blocks TPA-induced MAPK activation but has no effect on TPA-induced tyrosine phosphorylation. Our findings suggest that TPA-induced tyrosine phosphorylation of FAK and paxillin in human hepatoma cells is PKC dependent and requires the integrity of the cell cytoskeleton but is uncoupled to the signal transduction pathway of PKC leading to the translocation of PKC and MAPK activation.     

Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling

Protein kinase C (PKC) family members transduce an abundance of diverse intracellular signals. Here we address the role of spatial and temporal segregation in signal specificity by measuring the activity of endogenous PKC at defined intracellular locations in real time in live cells. We targeted a genetically encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter (CKAR) (Violin, J. D., Zhang, J., Tsien, R. Y., and Newton, A. C. (2003) J. Cell Biol. 161, 899-909), to the plasma membrane, Golgi, cytosol, mitochondria, or nucleus by fusing appropriate targeting sequences to the NH2 or COOH terminus of CKAR. Measuring the phosphorylation of the reporter in the presence of PKC inhibitors, activators, and/or phosphatase inhibitors shows that activity at each region is under differential control by phosphatase activity; nuclear activity is completely suppressed by phosphatases, whereas membrane-associated activity is the least suppressed by phosphatases. UTP stimulation of endogenous P2Y receptors in COS 7 cells reveals spatiotemporally divergent PKC responses. Imaging the second messengers Ca2+ and diacylglycerol (DAG) reveal that PKC activity at each location is driven by an initial spike in Ca2+, followed by location-specific diacylglycerol generation. In response to UTP, phosphorylation of GolgiCKAR was sustained the longest, driven by the persistence of DAG, whereas phosphorylation of CytoCKAR was of the shortest duration, driven by high phosphatase activity. Our data reveal that the magnitude and duration of PKC signaling is location-specific and controlled by the level of phosphatase activity and persistence of DAG at each location.     

Diacylglycerol kinase in plasma membranes from wheat.

Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg2+ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl2. The Mg2+ requirement could be substituted only partially by Mn2+ and not at all by Ca2+. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.     

IL-3-induced activation of protein kinases in the mast cell/megakaryocyte R6-XE.4 line

A role for second messenger-regulated protein kinases in the early post-IL-3 receptor signal transduction pathway was investigated in the mast cell/megakaryocyte line R6-XE.4. The activity of the calcium- and phospholipid-dependent protein kinase C (PKC) was assessed by the ability of the enzyme to phosphorylate histone H1 in the presence of calcium, diacylglycerol, and phosphatidylserine or after proteolytic activation of PKC with trypsin. In high serum-supplemented cells, but not in cells that were preincubated in serum-deficient media for 6 h, subsequent treatment for 15 min with synthetic IL-3 (10 micrograms/ml) caused up to a sixfold increase in the calcium- and lipid-stimulated histone H1 phosphorylating activity of particulate-associated PKC after fractionation on MonoQ. However, there was no corresponding reduction of cytosolic PKC activity. Therefore, IL-3 appeared to modify the activity of preexisting membrane-associated PKC rather than eliciting its recruitment from the cytoplasm in R6-XE.4 cells. This was in contrast to the situation with FDC-P1 cells, where IL-3 induced PKC translocation. IL-3 also stimulated a cytosolic protein kinase that phosphorylated a synthetic peptide patterned after a phosphorylation site in ribosomal protein S6, but this IL did not alter the activity of cAMP-dependent protein kinase.