p38 MAPK信号传导通路

丝裂原活化蛋白激酶（ｍｉｔｏｇｅｎ－ａｃｔｉｖａｔｅｄｐｏｒｏｔｅｉｎｋｉｎａｓｅ,ＭＡＰＫ）介导了生长、发育,分裂,死亡,以及细胞间的功能同步等多种细胞生理功能,在哺乳动物细胞中已发现和克隆了ＥＲＫ、ＪＮＫ／ＳＡＰＫ,ＥＲＫ５／ＢＭＫ１和ｐ３８／ＲＫ四个ＭＡＰＫ亚族,这些新的ＭＡＰＫ介导了物理,化学反激,细菌产物,炎性细胞因子等多种刺激引起的细胞反应,ｐ３８亚族至少包括ｐ３８（α）,ｐ３８β,ｐ     

大鼠脑内远位触液神经元p38丝裂原活化蛋白激酶的分布及在噪声应激时的表达

目的：观察脑内远位触液神经元内p-p38丝裂原活化蛋白激酶（MAPK）的分布及其在噪声应激时的表达。方法：用霍乱毒素亚单位B与辣根过氧化物酶复合物（CB-HRP）标记和免疫组织化学相结合的双重标记技术．观察SD大鼠脑实质内远位触液神经元中p-p38MAPK的分布：进一步制作噪声应激动物模型,观察噪声应激后该类神经元中p-p38MAPK的表达变化。结果：在脑干的特定部位恒定出现被CB-HRP标记的两组神经细胞簇,其他脑区未见CB-HRP标记神经细胞簇。不予应激刺激,该细胞簇内仅有个别神经元见有CB-HRP／p—p38MAPK;噪声应激刺激1d时,上述特定部位细胞簇的CB-HRP／p-p38MAPK双重标记神经元数目没有明显变化;噪音应激刺激5d时,CB-HRP／p—p38MAPK双重标记神经元数目较对照组显著增多（P〈0．05）;噪音应激刺激10d时CB-HRP／p—p38MAPK双重标记神经元数目较对照组显著增多（P〈0．05）;噪音应激刺激20d时,CB-HRP／p—p38MAPK双重标记神经元数目较对照组显著增多（P〈0．01）：结论：在脑干特定部位恒定存在的两组被CBHRP标记的细胞团为远位触液神经元,其中少数触液神经元有p-p38MAPK表达,且当给予动物噪声应激刺激时,p-p38MAPK免疫阳性神经元和CB-HRP／p—p38MAPK双重标记神经元数量显著增加,提示脑实质内的这种远位触液神经元中的P—p38MAPK可能参与了机体对噪声应激的信息传递或调控,其作用随应激天数增加而日趋增强．     

p38 MAPK as a signal transduction component of heavy metals stress in <Emphasis Type="Italic">Euglena gracilis</Emphasis>

Living organisms are subject to stress, and among these stressors, heavy metals exposure triggers accumulation of sulfur metabolites. Among these metabolites, glutathione and phytochelatins are found in several organisms, such as Euglena gracilis. Pre-exposing E. gracilis to low concentrations of Hg²⁺ generates a population with resistance to even 0.2 mM Cd²⁺, and this resistance relies partly on phytochelatins. p38 MAPK is stimulated by stress and is involved in apoptotic as well as survival mechanisms. In this study, we explored its participation in heavy metal-induced stress and its possible role in sulfur metabolite accumulation. We found that about 51% of the E. gracilis pretreated with Hg²⁺ becomes resistant to Cd²⁺ and proliferates despite the presence of this metal. The accumulation of the sulfur metabolites γ-glu-cys, glutathione and phytochelatin 2 displayed cyclic patterns that were disturbed by a challenge with Cd²⁺. We observed a p38 MAPK-like activity that was stimulated by acute or chronic heavy metal exposure, and its inhibition by SB203580 slightly diminished the accumulation of sulfur compounds. p38 MAPK inhibition also affected basal levels of glutathione in either pretreated or control cells. Thus, it appears that p38 MAPK mediates redox stress component of the signal pathway induced by heavy metals.     

The AtMKK3 pathway mediates ABA and salt signaling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Mitogen-activated protein (MAP) kinases cascades mediate cellular responses to a great variety of different extracellular signals in plants. Activation of a MAP kinase occurs after phosphorylation by an upstream dual-specificity protein kinase, known as a MAP kinase kinase. However, only a few of the MAPK kinases in Arabidopsis have been investigated. An active AtMKK3, 35S:AtMPK1, 35S:AtMPK2, and 35S:AtMPK3 constructs were built and their transformed plants were generated. The kinase activity of AtMPK1 or AtMPK2 was stimulated by active AtMKK3 in transient analysis of tobacco leaves. Coimmunoprecipitation experiments indicated interaction between AtMKK3 and AtMPK1 or AtMPK2 in the coexpressed tissues of AtMKK3 and AtMPK1 or AtMKK3 and AtMPK2. RT-PCR analysis showed that AtMKK3 and AtMPK1, or AtMKK3 and AtMPK2 were co-expressed in diverse plant tissues. Plants overexpressing AtMKK3 exhibited an enhanced tolerance to salt and were more sensitive to ABA. Plants overexpressing AtMPK1 or AtMPK2 were also more sensitive to ABA. AtMPK1 or AtMPK2 can be activated by cold, salt, and ABA. AtMKK3, AtMPK1, and AtMPK2 genes were induced by ABA or stress treatments. All these data indicated that the ABA signal transmitted to a MAPK kinase signaling cascade and could be amplified through MAP kinase1 or MAP kinase2 for increasing salt stress tolerance in Arabidopsis.     

Inhibition of P38 MAPK Reduces Tumor Conditioned Medium-Induced Angiogenesis in Co-Cultured Human Umbilical Vein Endothelial Cells and Fibroblasts

Tumor conditioned medium (CM) has been widely used to stimulate endothelial cells to form capillary-like structures in in vitro angiogenesis models. We report herein the effect of HT1080 and A549 CM after they were mixed with microvascular endothelial cells medium-2 (EGM-2) on angiogenesis in human umbilical vein endothelial cells (HUVECs). Both HT1080 and A549 CM decreased HUVEC proliferation, to different extents. While A549 CM significantly increased capillary-like structure formation in a co-culture system, no effect of HT1080 was apparent. Inhibition of p38 mitogen-activated protein kinase (MAPK) blocked both basal and A549 CM induced capillary-like structure formation, but inhibition of extracellular signal-regulated kinases (ERK) and that of c-Jun N-terminal protein kinases (JNK) MAPK had no such effect. Activation of ERK MAPK was inhibited by both CMs, whereas p38 MAPK was inactivated by HT1080 and activated by A549 CM and a control. Neither CM had an effect on JNK MAPK. The results suggest that p38 MAPK played a critical role in capillary-like structure formation in the co-culture, partly via promotion of apoptosis in HUVECs.     

beta-Amyloid peptide induces formation of actin stress fibers through p38 mitogen-activated protein kinase

Based on the critical role of actin in the maintenance of synaptic function, we examined whether expression of familial beta-amyloid precursor protein APP-V642I (IAPP) or mutant presenilin-1 L286V (mPS1) affects actin polymerization in rat septal neuronal cells. Expression of either IAPP or mPS1 but not wild-type amyloid precursor protein or presenilin-1induced formation of actin stress fibers in SN1 cells, a septal neuronal cell line. Treatment with beta-amyloid (Abeta) peptide also caused formation of actin stress fibers in SN1 cells and primary cultured hippocampal neurons. Treatment with a gamma-secretase inhibitor completely blocked formation of actin stress fibers, indicating that overproduction of Abeta peptide induces actin stress fibers. Because activation of the p38 mitogen-activated protein kinase (p38MAPK)-mitogen-associated protein kinase-associated protein kinase (MAPKAPK)-2-heat-shock protein 27 signaling pathway mediates actin polymerization, we explored whether Abeta peptide activates p38MAPK and MAPKAPK-2. Expression of IAPP or mPS1 induced activation of p38MAPK and MAPKAPK-2. Treatment with a p38MAPK inhibitor completely inhibited formation of actin stress fibers mediated by Abeta peptide, IAPP or mPS1. Moreover, treatment with a gamma-secretase inhibitor completely blocked activation of p38MAPK and MAPKAPK-2. In summary, our data suggest that overproduction of Abeta peptide induces formation of actin stress fibers through activation of the p38MAPK signaling pathway in septal neuronal cells.     

Isolation of active yeast telomerase protein Est3p and investigation of its dimerization <Emphasis Type="Italic">in vitro</Emphasis>

In this study we proposed a method for isolation of Est3p modified with various affinity tags, which is applicable for structural and functional studies, and investigated homo- and heterodimer formation with various recombinant forms of Est3p.     

Cell type-specific activation of p38 MAPK in the brain regions of hypoxic preconditioned mice

Activation of p38 mitogen-activated protein kinase (p38 MAPK) has been implicated as a mechanism of ischemia/hypoxia-induced cerebral injury. The current study was designed to explore the involvement of p38 MAPK in the development of cerebral hypoxic preconditioning (HPC) by observing the changes in dual phosphorylation (p-p38 MAPK) at threonine180 and tyrosine182 sites, protein expression, and cellular distribution of p-p38 MAPK in the brain of HPC mice. We found that the p-p38 MAPK levels, not protein expression, increased significantly (p < 0.05) in the regions of frontal cortex, hippocampus, and hypothalamus of mice in response to repetitive hypoxic exposure (H1–H6, n = 6 for each group) when compared to values of the control normoxic group (H0, n = 6) using Western blot analysis. Similar results were also confirmed by an immunostaining study of the p-p38 MAPK location in the frontal cortex, hippocampus, and hypothalamus of mice from HPC groups. To further define the cell type of p-p38 MAPK positive cells, we used a double-labeled immunofluorescent staining method to co-localize p-p38 MAPK with neurofilaments heavy chain (NF-H, neuron-specific marker), S100 (astrocyte-specific marker), and CD11b (microglia-specific maker), respectively. We found that the increased p-p38 MAPK occurred in microglia of cortex and hippocampus, as well as in neurons of hypothalamus of HPC mice. These results suggest that the cell type-specific activation of p38 MAPK in the specific brain regions might contribute to the development of cerebral HPC mechanism in mice.     

Baculovirus <Emphasis Type="Italic">per os</Emphasis> infectivity factors are involved in HearNPV ODVs infection of HzAM1 cells <Emphasis Type="Italic">in vitro</Emphasis>

Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-1, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry. Foundation items: National Nature Science Foundations of China (30325002, 30470075); National Basic Research Priorities Program of China (2003CB1140).     

p38丝裂原素激活的蛋白激酶在调节低氧诱导人内皮细胞分泌血管内皮生长因子过程中的作用

血管内皮细胞中血管内皮生长因子(vascular endothelial growthfactor,VEGF)的合成增加在促进血管新生的过程中起着非常重要的作用.然而低氧诱导VEGF分泌的细胞内信号转导机制还不是很清楚.人脐静脉内皮细胞系(ECV304)在低氧或常氧的状态下培养12～24 h后分别用实时定量PCR和Western blot的方法来检测VEGF mRNA的表达及ERK1/2和p38激酶的磷酸化水平.分泌到培养液中的VEGF蛋白用酶联免疫吸附(ELISA)的方法来检测.业已报道,ERK的抑制剂PD98059能够抑制低氧诱导的VEGF基因的表达,根据这个报道,我们发现在低氧情况下,ECV304细胞的ERK1/2磷酸化水平增高以及VEGF的合成增加等这些变化也能被PD98059所抑制.本次实验的新发现是p38激酶的激活在低氧诱导VEGF合成增加中的作用.p38激酶的抑制剂SB202190能抑制低氧诱导的VEGF合成增加.这些数据首次直接证实了p38激酶在低氧诱导人内皮细胞分泌VEGF增加过程中的作用.     

Monomeric and dimeric models of ERK2 in conjunction with studies on cellular localization,nuclear translocation,and <Emphasis Type="Italic">in vitro</Emphasis> analysis

Extracellular signal-regulated protein kinase 2 (ERK2) plays many vital roles in cellular signal regulation. Phosphorylation of ERK2 leads to propagation and execution of various extracellular stimuli, which influence cellular responses to stress. The final response of the ERK2 signaling pathway is determined by localization and duration of active ERK2 at specific target cell compartments through protein-protein interactions of ERK2 with various cytoplasmic and nuclear substrates, scaffold proteins, and anchoring counterparts. In this respect, dimerization of phosphorylated ERK2 has been suggested to be a part of crucial regulating mechanism in various protein-protein interactions. After the report of putative dimeric structure of active ERK2 (Canagarajah et al., 1997), dimeric model was employed to explain many in vivo and in vitro experimental results. But more recently, many reports have been presented questioning the validity of dimer hypothesis of active ERK2. In this review, we summarize the various in vitro and in vivo studies concerning the Monomeric or the dimeric forms of ERK2 and the validity of the dimer hypothesis.     

A Model to Induce Low Temperature Trauma for <Emphasis Type="Italic">in vitro</Emphasis> Astrogliosis Study

Astrogliosis is an inevitable and rapid response of astrocytes to physical, chemical and pathological injuries. To study astrogliosis, we developed a reproducible in vitro model in which low temperature injury to cultured astrocytes could be induced by placing the culture dish onto a copper pipe pre-cooled by liquid nitrogen. Using this model, the relationship between the temperature decline and the severity of cellular damage was analyzed. An increase in the expression of some known injury-related proteins, such as glial fibrillary acidic protein (GFAP), immediate early response genes (IEGs), and heat shock proteins 70 (HSP70), was demonstrated in astrocytes after low temperature trauma. With the use of this low temperature trauma model, the flexibility in the temperature control and injury area may allow researchers to evaluate cryotherapy and cryosurgery, which could be applicable to future development of quality health care.Special issue dedicated to Lawrence F. Eng.     

促分裂原激活的蛋白激酶(MAPK)信号传导通路的研究进展

MAPK信号传导通路在真核生物细胞的生化和分化、细胞周期调节和细胞凋亡过程中发挥着重要的作用。生物化学研究和分子生物学鉴定表明：在酵母和哺乳动物细胞中MAPK信号传导通路都有一个保守的三组分激活模件,该模件内的激酶引发了一系列的磷酸化级联反应。了解MAPK信号传导通路的组成部分、调控方式和作用机制,有助于对因信号传导通路的调节失控而引起的疾病进行预防和治疗。     

Molecular analysis of the<Emphasis Type="Italic"> CRINKLY4</Emphasis> gene family in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The maize (Zea mays L.) CRINKLY4 (CR4) gene encodes a serine/threonine receptor-like kinase that controls an array of developmental processes in the plant and endosperm. The Arabidopsis thaliana (L.) Heynh. genome encodes an ortholog of CR4, ACR4, and four CRINKLY4-RELATED (CRR) proteins: AtCRR1, AtCRR2, AtCRR3 and AtCRK1. The available genome sequence of rice (Oryza sativa L.) encodes a CR4 ortholog, OsCR4, and four CRR proteins: OsCRR1, OsCRR2, OsCRR3 and OsCRR4, not necessarily orthologous to the Arabidopsis CRRs. A phylogenetic study showed that AtCRR1 and AtCRR2 form a clade closest to the CR4 group while all the other CRRs form a separate cluster. The five Arabidopsis genes are differentially expressed in various tissues. A construct formed by fusion of the ACR4 promoter and the GUS reporter, ACR4::GUS, is expressed primarily in developing tissues of the shoot. The ACR4 cytoplasmic domain functions in vitro as a serine/threonine kinase, while the AtCRR1 and AtCRR2 kinases are not active. The ability of ACR4 to phosphorylate AtCRR2 suggests that they might function in the same signal transduction pathway. T-DNA insertions were obtained in ACR4, AtCRR1, AtCRR2, AtCRR3 and AtCRK1. Mutations in acr4 show a phenotype restricted to the integuments and seed coat, suggesting that Arabidopsis might contain a redundant function that is lacking in maize. The lack of obvious mutant phenotypes in the crr mutants indicates they are not required for the hypothetical redundant function.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Pisum sativum in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

Six pea (Pisum sativum L.) cultivars (Adept, Komet, Lantra, Olivin, Oskar, Tyrkys) were transformed via Agrobacterium tumefaciens strain EHA105 with pBIN19 plasmid carrying reporter uidA (β-glucuronidase, GUS, containing potato ST-LS1 intron) gene under the CaMV 35S promoter, and selectable marker gene nptII (neomycin phosphotransferase II) under the nos promoter. Two regeneration systems were used: continual shoot proliferation from axillary buds of cotyledonary node in vitro, and in vivo plant regeneration from imbibed germinating seed with removed testa and one cotyledon. The penetration of Agrobacterium into explants during co-cultivation was supported by sonication or vacuum infiltration treatment. The selection of putative transformants in both regeneration systems carried out on media with 100 mg dm⁻³ kanamycin. The presence of introduced genes was verified histochemically (GUS assay) and by means of PCR and Southern blot analysis in T₀ putative transformants and their seed progenies (T₁ to T₃ generations). Both methods, but largely in vivo approach showed to be genotype independent, resulting in efficient and reliable transformation system for pea. The in vivo approach has in addition also benefit of time and money saving, since transgenic plants are obtained in much shorter time. All tested T₀ – T₃ plants were morphologically normal and fertile.This research was supported by the National Agency for Agricultural Research (grants No. QE 0046 and QF 3072) and Ministry of Education of the Czech Republic (grant No. ME 433).