肺炎支原体P1蛋白的研究现状

P1蛋白是肺炎支原体的一种与黏附有关的跨膜蛋白,它只有正确定位于肺炎支原体顶端结构才能介导其黏附作用;P1蛋白结构基因的多态性是构成肺炎支原体抗原多态性的基础;P1蛋白本身也是一种重要的免疫原,可刺激机体产生强烈的免疫应答。因此,探讨P1蛋白基因结构与功能将有助于肺炎支原体的致病机制及其感染的诊断和预防等方面的研究。     

肺炎支原体实验室检测方法研究进展

肺炎支原体(Mycoplosma pneumonia,MP)为人类非典型肺炎的病原体,是引起呼吸道感染的重要病原体。但是支原体肺炎与其他病原体感染的肺炎,在临床症状、影像学上并无特异性差别,且其对一般治疗肺炎、上呼吸道感染的药物有耐药性,因此肺炎支原体及时、准确的实验室检测对于支原体肺炎的诊断治疗显得尤为重要。目前MP的实验室检测方法不断推陈出新,但各种方法均有其优势与不足,临床可选择两种不同的方法同时检测。比如:血清学抗体的检测结合MP快速培养药敏的方法;血清学抗体的检测结合PCR的方法,不同方法相互补充为临床的早期诊断、治疗提供依据。而MP药敏试验的检测和耐药机制的研究对于临床用药方案的选择,减少耐药株的产生和流行具有重要意义。     

Dickkopf相关蛋白1抑制肺炎支原体P1-C诱导小鼠肺上皮细胞过度分泌MUC5AC

肺炎支原体(Mycoplasma pneumoniae)是儿童和成人最常见的呼吸道感染病原体。临床观察肺炎支原体感染会引起呼吸道黏液大量分泌,给患者呼吸造成困难,已有研究表明肺炎支原体感染会引起大量黏蛋白5AC (mucin 5AC,MUC5AC)的分泌。肺炎支原体P1黏附素通过介导病原体与宿主细胞的黏附在肺炎支原体感染的发病机制中发挥重要作用,其中P1的C-末端残基(P1-C)具有免疫原性。本研究探讨了Wnt(Wingless,Wnt)/β-catenin信号通路抑制因子Dickkopf-1(Dickkopf-1, DKK1)在肺炎支原体P1-C诱导的肺上皮细胞分泌黏蛋白MUC5AC的分子机制。利用扫描电镜(scanning electron microscope, SEM)、苏木精-伊红(hematoxylin-eosin, HE)染色观察肺炎支原体P1-C对小鼠肺上皮细胞(mouse airway epithelial cells, MAECs)黏液分泌的影响;利用蛋白芯片技术检测肺炎支原体P1-C对小鼠气道上皮细胞炎症因子分泌及对相关信号通路的富集分析;采用糖原染色(perio...     

肺炎支原体P1重组蛋白的提取纯化及应用研究

提取纯化肺炎支原体(Mycoplasma pneumoniae,Mp)P1重组蛋白,建立酶联免疫吸附实验(ELISA)的方法,协助临床肺炎支原体感染的诊断。以GST融合蛋白层析柱提取、纯化Mp P1重组蛋白做抗原,以全肺炎支原体菌体成分做抗原对照,建立间接ELISA实验方法,检测40份正常献血者血清标本和51份疑似MP感染临床血清标本的IgG抗体。重组蛋白经SDS-PAGE可见诱导表达的样品在分子量大约59 ku处有明显条带,经Western blotting可与肺炎支原体免疫血清发生反应。ELISA实验检测51份临床标本,由P1重组蛋白抗原检测阳性31份,阳性率为60.78%。Mp检测阳性20份,阳性率为39.22%。实验精确度检测阳性混合血清的变异系数(CV值)为5.40%,阴性混合血清变异系数为1.10%。用Mp P1重组蛋白抗原建立的ELISA检测方法,其敏感性高于全肺炎支原体抗原,可用于临床肺炎支原体感染的诊断。     

肺炎支原体主要黏附蛋白的研究进展

肺炎支原体(Mycoplasma pneumoniae,Mp)可引起呼吸道感染,是儿童社区获得性肺炎的常见致病菌,并且可导致多系统并发症。Mp尖端的黏附蛋白复合体与肺纤毛上皮细胞的黏附过程是其主要致病机制。P1、P30、P116等蛋白作为黏附复合体的主要成分,在感染过程中起到了关键作用。在缺失这些蛋白的突变株中,细胞结构异常,黏附能力、运动速度、致病性均表现出不同程度的下降。由于Mp生长缓慢,对营养要求苛刻,常规微生物分离培养不适用于临床快速诊断,目前亚洲地区Mp耐药率居高不下,Mp感染后又无特异性临床症状,使得临床工作中Mp感染的诊治常被延误。重组蛋白表达技术的出现,使得Mp主要蛋白可以应用在Mp诊断试剂盒和疫苗研发中,本文针对Mp主要黏附蛋白的基因结构、致病机制及其应用方面的最新研究进展进行综述。     

密码子优化的肺炎支原体P1蛋白在大肠杆菌中的克隆表达

目的:获得密码子优化的肺炎支原体P1黏附蛋白优势表位抗原基因,并在大肠杆菌中表达,为临床诊断试剂和疫苗研制打下基础。方法:采用生物信息学分析肺炎支原体P1蛋白的抗原表位,筛选特异性P1蛋白优势表位区;采用大肠杆菌优势密码子,设计上述P1蛋白优势表位基因序列;采用退火PCR技术合成上述基因,并利用载体pGEX-4T-2实现P1优势表位抗原在大肠杆菌中的表达;采用ELISA法对纯化的P1抗原活性进行测定。结果:肺炎支原体P1蛋白特异性抗原表位主要位于1154~1521 aa,获得的P1优化密码子基因平行突变37个稀有密码子和2个终止密码子;在大肠杆菌中表达的GST-P1融合蛋白的相对分子质量为65.9×103,纯化后重组抗原能与肺炎支原体感染者血清发生特异性的免疫反应。结论:采用密码子优化基因合成技术实现了肺炎支原体P1优势表位抗原在大肠杆菌中的高效表达,为肺炎支原体感染的诊断试剂研究提供了重要参考。     

肺炎支原体感染鼠P1特异抗原的动态检测

通过实验动物模型探讨肺炎支原体感染动物肺泡灌洗液中特异抗原检出率的动态变化,为肺炎支原体感染的临床诊断提供理论依据。小鼠经鼻自然感染肺炎支原体,分别采集感染后不同时间点小鼠的支气管灌洗液,应用量子点标记肺炎支原体P1蛋白抗体,直接免疫荧光法检测感染鼠肺泡灌洗液中肺炎支原体P1特异抗原,同时通过PCR检测肺组织肺炎支原体DNA及肺组织病理切片观察肺部炎性变化确定小鼠感染。结果显示,感染鼠肺炎支原体特异抗原在感染后第3天检出阳性率为75%,第7天达高峰为83%,之后随病程延长,抗原检测的阳性率逐渐下降,在感染后第14、21天检出阳性率分别为58%和25%。肺炎支原体特异抗原在感染早期检出率高。应用量子点标记肺炎支原体P1蛋白抗体,直接免疫荧光法检测肺炎支原体特异抗原可应用于肺炎支原体感染的早期诊断。     

儿科呼吸道疾病中肺炎支原体感染的结果分析

目的探讨儿科肺炎支原体（MP）感染特点,辅助临床医师早期诊断,合理用药。方法测定我院一年来2013例儿科呼吸道疾病患儿的肺炎支原体抗体（IgM）。结果2013例呼吸道感染患儿,检出肺炎支原体抗体（IgM）阳性者769例,占38.2%,769例阳性分别表现为肺炎369例（48%）,支气管炎238例（31%）,咽炎92例（12%）,哮喘70例（9%）。其中,肺炎组与各组相比较,具有统计学意义。MP IgM感染的检出率明显高于其他各组（P〈0.01）。结论MP感染是患儿不可忽视的病原体,检测患儿血清MP抗体能够及早诊断,指导治疗。     

肺炎支原体表面黏附蛋白的研究进展

肺炎支原体感染可引发非典型肺炎和多种上呼吸道疾病。其致病过程主要由表面黏附蛋白介导完成。本文综述了肺炎支原体表面黏附蛋白的研究进展。     

猪肺炎支原体及其他支原体黏附因子的研究进展

猪肺炎支原体能够引起猪支原体肺炎,其黏附因子在致病过程中起重要作用。本文综合国内外猪肺炎支原体黏附因子的研究进展,并与其他支原体的黏附因子进行了比较讨论,从而为猪肺炎支原体致病机理的进一步研究及该病的防制提供新思路。     

Myelin protein zero/P0 phosphorylation and function require an adaptor protein linking it to RACK1 and PKC alpha

Point mutations in the cytoplasmic domain of myelin protein zero (P0; the major myelin protein in the peripheral nervous system) that alter a protein kinase Calpha (PKCalpha) substrate motif (198HRSTK201) or alter serines 199 and/or 204 eliminate P0-mediated adhesion. Mutation in the PKCalpha substrate motif (R198S) also causes a form of inherited peripheral neuropathy (Charcot Marie Tooth disease [CMT] 1B), indicating that PKCalpha-mediated phosphorylation of P0 is important for myelination. We have now identified a 65-kD adaptor protein that links P0 with the receptor for activated C kinase 1 (RACK1). The interaction of p65 with P0 maps to residues 179-197 within the cytoplasmic tail of P0. Mutations or deletions that abolish p65 binding reduce P0 phosphorylation and adhesion, which can be rescued by the substitution of serines 199 and 204 with glutamic acid. A mutation in the p65-binding sequence G184R occurs in two families with CMT, and mutation of this residue results in the loss of both p65 binding and adhesion function.     

Role of P30 in replication and spread of TMV

The P30 movement protein (MP) of tobacco mosaic virus is essential for distribution of sites of replication within infected cells and for cell–cell spread of infection. MP is an integral membrane protein and in early and mid-stages of infection causes severe disruption of the cortical endoplasmic reticulum (ER). MP also associates with microtubules, and in late stages is targeted for degradation by the 26S proteosome. During these stages, the ER regains its normal pre-infection configuration. Viral RNA is associated with ER and microtubules in the presence of MP. The MP is phosphorylated and mutation of the phosphorylated amino acid reduced association of MP with the ER, plasmodesmata, and microtubules, and altered the stability of the MP. The nature of the association of MP with vRNA and ER and microtubules, and the role of phosphorylation of MP in each of these functions, if any, remains to be determined.     

MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily,joins the list of ligands of galectin-3

Background  

Although MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator.     

Regulation of cellular proliferation, differentiation and cell death by activated Raf

ERK signaling regulates focal adhesion disassembly during cell movement, and increased ERK signaling frequently contributes to enhanced motility of human tumor cells. We previously found that the ERK scaffold MEK Partner 1 (MP1) is required for focal adhesion disassembly in fibroblasts. Here we test the hypothesis that MP1-dependent ERK signaling regulates motility of DU145 prostate cancer cells. We find that MP1 is required for motility on fibronectin, but not for motility stimulated by serum or EGF. Surprisingly, MP1 appears not to function through its known binding partners MEK1 or PAK1, suggesting the existence of a novel pathway by which MP1 can regulate motility on fibronectin. MP1 may function by regulating the stability or expression of paxillin, a key regulator of motility.     

The MEK1 scaffolding protein MP1 regulates cell spreading by integrating PAK1 and Rho signals

How the extracellular signal-regulated kinase (ERK) cascade regulates diverse cellular functions, including cell proliferation, survival, and motility, in a context-dependent manner remains poorly understood. Compelling evidence indicates that scaffolding molecules function in yeast to channel specific signals through common components to appropriate targets. Although a number of putative ERK scaffolding proteins have been identified in mammalian systems, none has been linked to a specific biological response. Here we show that the putative scaffold protein MEK partner 1 (MP1) and its partner p14 regulate PAK1-dependent ERK activation during adhesion and cell spreading but are not required for ERK activation by platelet-derived growth factor. MP1 associates with active but not inactive PAK1 and controls PAK1 phosphorylation of MEK1. Our data further show that MP1, p14, and MEK1 serve to inhibit Rho/Rho kinase functions necessary for the turnover of adhesion structures and cell spreading and reveal a signal-channeling function for a MEK1/ERK scaffold in orchestrating cytoskeletal rearrangements important for cell motility.     

Identification and characterization of mechanism of action of P61-E7, a novel phosphine catalysis-based inhibitor of geranylgeranyltransferase-I

Small molecule inhibitors of protein geranylgeranyltransferase-I (GGTase-I) provide a promising type of anticancer drugs. Here, we first report the identification of a novel tetrahydropyridine scaffold compound, P61-E7, and define effects of this compound on pancreatic cancer cells. P61-E7 was identified from a library of allenoate-derived compounds made through phosphine-catalyzed annulation reactions. P61-E7 inhibits protein geranylgeranylation and blocks membrane association of geranylgeranylated proteins. P61-E7 is effective at inhibiting both cell proliferation and cell cycle progression, and it induces high p21(CIP1/WAF1) level in human cancer cells. P61-E7 also increases p27(Kip1) protein level and inhibits phosphorylation of p27(Kip1) on Thr187. We also report that P61-E7 treatment of Panc-1 cells causes cell rounding, disrupts actin cytoskeleton organization, abolishes focal adhesion assembly and inhibits anchorage independent growth. Because the cellular effects observed pointed to the involvement of RhoA, a geranylgeranylated small GTPase protein shown to influence a number of cellular processes including actin stress fiber organization, cell adhesion and cell proliferation, we have evaluated the significance of the inhibition of RhoA geranylgeranylation on the cellular effects of inhibitors of GGTase-I (GGTIs). Stable expression of farnesylated RhoA mutant (RhoA-F) results in partial resistance to the anti-proliferative effect of P61-E7 and prevents induction of p21(CIP1/WAF1) and p27(Kip1) by P61-E7 in Panc-1 cells. Moreover, stable expression of RhoA-F rescues Panc-1 cells from cell rounding and inhibition of focal adhesion formation caused by P61-E7. Taken together, these findings suggest that P61-E7 is a promising GGTI compound and that RhoA is an important target of P61-E7 in Panc-1 pancreatic cancer cells.     

Optimal T cell responses to Cryptococcus neoformans mannoprotein are dependent on recognition of conjugated carbohydrates by mannose receptors

Cryptococcosis is a leading cause of death among individuals with compromised T cell function. Soluble Cryptococcus neoformans mannoproteins (MP) have emerged as promising vaccine candidates due to their capacity to elicit delayed-type hypersensitivity and Th type 1-like cytokines, both critical to the clearance of this pathogenic yeast. In this study, the mechanisms responsible for the potent immunostimulatory properties of MP were explored. Using Chinese hamster ovary cells expressing human macrophage mannose receptor (MMR), we determined that MP is a MMR ligand. Functionally, competitive blockade of multilectin mannose receptors (MR) on APCs diminished MP-dependent stimulation of primary T cells from immunized mice and the MP-reactive CD4(+) T cell hybridoma, P1D6, by 72 and 99%, respectively. Removal of O-linked saccharides from MP by beta-elimination inhibited MP-dependent stimulation of P1D6 and primary T cells by 89 and 90%, respectively. In addition, MP-dependent stimulation of P1D6 was abrogated after digestion with proteinase K, suggesting the protein core of MP contributed the antigenic moiety presented by APC. Stimulation of P1D6 by MP also was abolished using APC obtained from invariant chain-deficient mice, demonstrating Ag presentation was MHC class II restricted. Our data suggest that MP is a ligand for the MMR and that T cell stimulation is functionally inhibited either by competitive blockade of MR or by removal of carbohydrate residues critical for recognition. The demonstration that efficient T cell responses to MP require recognition of terminal mannose groups by MMR provides both a molecular basis for the immunogenicity of cryptococcal MP and support for vaccination strategies that target MR.