p62 Is a Common Component of Cytoplasmic Inclusions in Protein Aggregation Diseases

Exposure of cells to stress, particularly oxidative stress, leads to misfolding of proteins and, if they are not refolded or degraded, to cytoplasmic protein aggregates. Protein aggregates are characteristic features of a variety of chronic toxic and degenerative diseases, such as Mallory bodies (MBs) in hepatocytes in alcoholic and non-alcoholic steatohepatitis, neurofibrillary tangles in neurons in Alzheimer's, and Lewy bodies in Parkinson's disease. Using 2D gel electrophoresis and mass spectrometry, we identified p62 as a novel MB component. p62 and cytokeratins (CKs) are major MB constituents; HSP 70, HSP 25, and ubiquitinated CKs are also present. These proteins characterize MBs as a prototype of disease-associated cytoplasmic inclusions generated by stress-induced protein misfolding. As revealed by transfection of tissue culture cells overexpressed p62 did not induce aggregation of regular CK filaments but selectively bound to misfolded and ubiquitinated CKs. The general role of p62 in the cellular response to misfolded proteins was substantiated by detection of p62 in other cytoplasmic inclusions, such as neurofibrillary tangles, Lewy bodies, Rosenthal fibers, intracytoplasmic hyaline bodies in hepatocellular carcinoma, and alpha1-antitrypsin aggregates. The presence of p62 along with other stress proteins and ubiquitin in cytoplasmic inclusions indicates deposition as aggregates as a third line of defense against misfolded proteins in addition to refolding and degradation.     

The EspD Protein of Enterohemorrhagic Escherichia coli Is Required for the Formation of Bacterial Surface Appendages and Is Incorporated in the Cytoplasmic Membranes of Target Cells

The formation of EspA-containing surface appendages in pathogenic Escherichia coli strains, both enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. coli strains, is essential for critical events in the infective process, e.g., localized bacterial adherence to host cells with formation of microcolonies and induction of attaching and effacing lesions. It has been reported that EPEC mutants deficient in the production of EspD, which is encoded by the esp operon, are unable to accumulate actin underneath adherent bacteria but exhibit an attachment similar to that of the wild type. Here, we report the construction and characterization of an in-frame espD deletion mutant of the enterohemorrhagic E. coli (EHEC) strain EDL933. In contrast to what was observed in EPEC mutants, the EDL933 espD mutant not only lacked the capacity to accumulate actin but also exhibited an impaired attachment to HeLa cells. The synthesis of the EspD protein was also essential for the formation of EspA-containing filaments. Finally, localization studies demonstrated that the EspD protein is transferred to the cytoplasm and integrated into the cytoplasmic membranes of infected cells. These results help to elucidate the underlying molecular events in infections caused by EHEC.     

人GST-PTEN融合蛋白的原核表达和纯化

目的构建人第10号染色体上磷酸酶和张力蛋白同源缺失的基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)原核表达质粒,使其在原核细胞中高效表达并进行纯化。方法将全长片段插入原核表达载体pGEX-4T-1,构建重组子pGEX-4T-1-PTEN,转化BL-21感受态细胞。经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达PTEN融合蛋白的可诱导性表达,通过SDS-PAGE电泳、Western印迹分析证实蛋白表达的特异性。并用谷胱甘肽-S-转移酶(glutathione-Stransferase,GST)亲和层析对融合蛋白进行纯化。结果成功构建了原核表达载体pGEX-4T-1-PTEN,并将PTEN融合蛋白的成功表达,通过SDS-PAGE电泳、Western印迹分析,证实了蛋白表达的特异性。并对蛋白进行了纯化,获得了GST-PTEN融合蛋白的纯品。结论成功表达、纯化了GST-PTEN融合蛋白,为进一步研究PTEN蛋白的功能及其与其它功能性蛋白的相互作用研究奠定了基础。     

Induction of Interleukin-4 and Interleukin-5 Expression in Mast Cells Is Inhibited by Glucocorticoids

Inflammation in asthma and other allergic diseases is characterized by excessive production of immunoglobulin E (IgE) and the influx of leukocytes, especially eosinophils. Interleukin 4 (IL-4) and IL-5 are essential for IgE production and eosinophilia, respectively, and are produced by mast cells in allergic conditions, for which glucocorticoids are widely used therapeutically. We assessed the effect of glucocorticoids on IL-4 and IL-5 mRNA production by the RBL-2H3 cell line, an analog of mucosal mast cells. IL-4 and IL-5 mRNAs were induced by an antigen that is used to cross-link receptor bound IgE, by calcium ionophore, or by ionophore with phorbol ester and were markedly inhibited by dexamethasone. In cells activated with ionophore and phorbol ester, 10⁻⁶ M dexamethasone reduced the IL-4 and IL-5 mRNA levels to only 12.8 and 5.7%, respectively, of those in cells without dexamethasone, and 10⁻⁹ M dexamethasone caused reductions to 27 and 56%, respectively. Hydrocortisone at 10⁻⁶ and 10⁻⁷ M almost completely inhibited IL-4 and IL-5 mRNA production. Dexamethasone was markedly inhibitory even if it was added after the cells were activated, provided that it was present in the cultures for at least 1.5 h. These studies indicate that the expression of IL-4 and IL-5 mRNAs by mast cells is highly sensitive to glucocorticoids. The data suggest that these inhibitory effects may contribute to the clinical efficacy of glucocorticoids in the therapy of allergic diseases.     

Gla-Rich Protein Is a Novel Vitamin K-Dependent Protein Present in Serum That Accumulates at Sites of Pathological Calcifications

Mineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization.Extracellular matrix (ECM) calcification can be either a physiological or a pathological process depending on site and time of occurrence. Physiological ECM calcification is restricted to bone and to the hypertrophic zones of growth plate cartilage, whereas pathological or ectopic ECM calcification, defined as inappropriate biomineralization occurring in soft tissues and consisting of calcium phosphate salts that include hydroxyapatite, is an abnormal process that can occur virtually in any tissue of the body.¹ However, skin, kidney, tendons, and the cardiovascular system appear particularly prone to develop this pathology.²First considered to be a passive process occurring as a nonspecific response to tissue injury or necrosis, recent evidence now indicates that ECM calcification is a naturally occurring process that must be actively inhibited and starts to appear as soon as inhibitors are removed from the matrix.^1,3,4 In a healthy organism, cells appear to synthesize natural inhibitors of mineralization that prevent ectopic calcification, which initiates when disequilibrium occurs between expression of calcification inhibitors and enhancers, emphasizing the need for a tight regulation to prevent ectopic calcifications.Key genes known to be involved in the regulation of this complex process are those acting as calcification inhibitors such as matrix Gla protein (MGP), osteocalcin (BGP), bone sialoprotein (BSP), osteoprotegerin (Opg), and fetuin.^1,3 Among those, MGP, a vitamin K-dependent protein (VKD), is widely accepted as playing a pivotal role in preventing soft tissue calcification, local mineralization of the vascular wall,⁵ and more recently, skin elastic fiber mineralization in pseudoxanthoma elasticum (PXE)^6,7,8 and in scleroderma with and without calcinosis.⁹ It is also known that several factors, such as insufficient intake of vitamin K, mutations in the γ-carboxylase enzyme, and warfarin treatment, which can all induce arterial^10,11,12 and skin calcifications,^7,13,14,15 may act by reducing or abolishing γ-carboxylation of VKD proteins. Those pathologies have also been associated with a loss of MGP function, until now considered to be the central Gla protein for prevention of connective tissue mineralization, both in the vascular system and skin. Although many efforts have been made to understand the mechanisms controlling these abnormal calcifications, the existence of other potential, still unknown, calcification inhibitors has been suggested to explain some reported phenotypes and occurrences that are not completely justified by the presence or absence of MGP.^1,16,17We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), with an unprecedented high content of Gla residues and uncommonly high capacity to bind calcium, with orthologs in all taxonomic groups of vertebrates and highly conserved throughout evolution (78% identity between sturgeon and human GRP).¹⁸ GRP mRNA was found to be highly expressed in sturgeon cartilaginous tissues, and in rat skeletal tissues, both cartilage and bone, which invalidated the concept that this protein could be solely a specific marker for distal chondrocytes, as previously proposed by others.¹⁸ In this study we show, for the first time, that GRP is a circulating protein also expressed and accumulated in soft tissues like skin and vascular system of rats and humans and that it is clearly associated with calcification pathologies in these tissues, being highly accumulated at sites of ectopic mineral deposits. Furthermore, the extensive number of Gla residues (16 Gla residues in sturgeon and, by comparison, 15 in all mammals) and the absence of other identifiable functional domains, together with our in vivo and in vitro evidence for a high mineral binding affinity, strongly suggest that GRP might be a potent physiological modulator of soft tissue calcification, acting by directly influence mineral formation and or recruitment, and an important new player in the complexity of phenotypes involving connective tissue mineralization, whose mechanisms and regulatory pathways remain to be fully understood.     

链霉亲和素-自噬相关基因融合蛋白的原核表达及纯化

目的 构建链霉亲和素（streptavidin,SA）和自噬相关基因（Beclin 1）重组质粒,并对融合蛋白SA-Beclin 1进行表达和纯化.方法 利用基因重组技术将核心SA与Beclin 1序列连接,克隆人pQE80形成pQE80-SA-Beclin 1重组载体,IPTG诱导融合蛋白原核表达,镍亲和凝胶层析柱纯化融合蛋白,Western blot鉴定.结果 PCR成功扩增核心SA活性中心,酶切鉴定和测序均证实重组载体pQE80-SA-Beclin 1构建成功;IPTG诱导后SA-Beclin 1融合蛋白（含His标签）在大肠杆菌中高效表达,SDS-PAGE分析表达的融合蛋白以包涵体为主,经镍亲和凝胶层析柱纯化得到融合蛋白,Western blot鉴定其相对分子量约为72000kD,与预期相符.结论 本文成功构建了重组质粒并表达纯化了SA-Beclin 1融合蛋白,为进一步研究SA-Beclin 1的功能及临床应用奠定了基础.     

Nonneural Nuclear Inclusions of Androgen Receptor Protein in Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy is an X-linked motor neuronopathy caused by the expansion of an unstable CAG repeat in the coding region of the androgen receptor (AR) gene. Nuclear inclusions of the mutant AR protein have been shown to occur in the spinal motor neurons of spinal and bulbar muscular atrophy (Li M, Kobayashi Y, Merry D, Tanaka F, Doyu M, Hashizume Y, Fischbeck KH, Sobue G: Nuclear inclusions in spinal and bulbar muscular atrophy. Ann Neurol 1998 (in press)). In this study, we demonstrate the tissue-specific distribution, immunochemical features, and fine structure of nuclear inclusions of spinal and bulbar muscular atrophy. Nuclear inclusions were observed in affected spinal and brainstem motor neurons, but not in other, nonaffected neural tissues. Similar nuclear inclusions occurred in nonneural tissues including scrotal skin, dermis, kidney, heart, and testis, but not in the spleen, liver, and muscle. These inclusions had similar epitope features detectable by antibodies that recognize a small portion of the N-terminus of the AR protein only, and they were ubiquitinated. Electron microscopic immunohistochemistry showed dense aggregates of AR-positive granular material without limiting membrane, both in the neural and nonneural inclusions. These findings indicate that nuclear inclusions of AR protein are present in selected nonneural tissues as well as in neurons that degenerate in spinal and bulbar muscular atrophy, suggesting that a common mechanism underlies in the formation of neural and nonneural nuclear inclusions.     

人Nephrin -GFP融合蛋白真核表达载体的构建和鉴定

目的 构建人19号染色体长臂Nephrin基因和绿色荧光蛋白(green fluorescence protein,GFP)真核表达质粒,为进一步研究肾小球裂隙隔膜分子复合物构成与功能提供基础.方法 设计引物,扩增真核表达质粒pEGFP N3中的GFP基因片段,将其插入nephrin原核表达载体pcDNA3.1 nephrin V5-His,重组质粒经酶切鉴定后测序,并转染至COS-7细胞观察表达情况及生物学特性.结果 成功构建pcDNA3.1 nephrin-GFP重组质粒,并将Nephrin -GFP融合蛋白成功表达于COS-7细胞,进一步经交联实验证明Nephrin-GFP融合蛋白具有正确的细胞膜表达.结论 利用pEGFP N3和pcDNA3.1 nephrin V5-His可成功重组Nephrin-GFP表达质粒,为进一步研究肾小球裂隙隔膜分子复合物构成及其功能提供有利工具.     

CTLA-4-FasL融合分子的构建及其生物学特性的鉴定

文章通过特异的引物分别扩增出CTLA 4和FasL胞外区的cDNA ,将它们拼接后 ,克隆入真核表达载体pcDNA3 1( + )中 ,进行表达、纯化 ,获得CTLA 4 FasL融合蛋白。Westernblot分析显示了该融合蛋白具有CTLA4 胞外区和FasL胞外区的抗原性。体外试验表明 ,该融合蛋白可以结合Jurkat细胞表面的Fas受体和Raji细胞表面的B7分子 ,表明了该分子双特异性的特点。该融合蛋白能够直接诱导Jurkat细胞发生凋亡 ,且此凋亡效应伴随Raji细胞的参与而增强 ,初步证实了该分子的免疫抑制效应 ,从而为进一步研究该融合蛋白特性及应用奠定了基础。     

Protein Synthesis Is Required for Induction of Amnesia Elicited by Disruption of the Reconsolidation of Long-Term Memory

Studies on common snails previously trained to an associative skill consisting of rejecting a defined foodstuff addressed the effects of NMDA glutamate receptor antagonists (MK-801 and APV) and protein synthesis inhibitors (cycloheximide and anisomycin) on long-term memory reconsolidation processes. Injections of each of the study compounds before the reminding procedure 24 h after training were found to lead to impairment of the reproduction of the acquired skill, which lasted at least three weeks. Repeat training of these animals to reject the same foodstuff as used in the initial training did not lead to acquisition of the skill. However, simultaneous injections of a protein synthesis inhibitor and an NMDA receptor antagonist (MK-801 + cycloheximide or APV + anisomycin) did not impair the skill. In subsequent experiments, snails received cycloheximide at different times after exposure to MK-801/reminding. Administration of cycloheximide 3 and 6 h after MK-801/reminding led to the development of incomplete amnesia and repeat training of the animals led to rapid restoration of memory. Administration of cycloheximide 9 h after MK-801/reminding evoked the development of stable amnesia characterized by impairment of skill formation on repeat training. We propose that the mechanisms of amnesia induced by the NMDA glutamate receptor antagonist, by analogy with the mechanisms of other long-term adaptive rearrangements of the brain, depend on translation and can be suppressed by inhibitors of translation. The “time window” for the dependence of amnesia induction processes on the synthesis of protein molecules was 6–9 h after exposure to MK-801/reminding.     

Septin8红色荧光蛋白融合载体的构建及表达

目的构建在哺乳动物细胞中表达的septin8红色荧光蛋白融合表达载体（pmCherry—C2／septin8）,并观察其在细胞内的表达和定位情况。方法采用PCR的方法从人脑胶质细胞瘤eDNA文库中扩增获得septin8基因编码区序列,将其克隆至红色荧光蛋白载体pmCherry—C2上,重组质粒经PCR、酶切以及序列分析正确无误后转染NIH3T3细胞,并利用Westernblotting和细胞免疫化学技术分析重组蛋白在细胞内的表达和定位。结果重组pmCherry—C2／septin8融合蛋白在NIH3T3细胞中得到高量表达且主要分布于细胞浆。结论成功构建了pmCherry—C2／septin8融合表达载体,该载体能在哺乳动物细胞NIH3T3中表达,为进一步研究septin8细胞内生物学功能打下了良好的基础。     

Clearance of Pseudomonas aeruginosa from a Healthy Ocular Surface Involves Surfactant Protein D and Is Compromised by Bacterial Elastase in a Murine Null-Infection Model

Our previous studies showed that surfactant protein D (SP-D) is present in human tear fluid and that it can protect corneal epithelial cells against bacterial invasion. Here we developed a novel null-infection model to test the hypothesis that SP-D contributes to the clearance of viable Pseudomonas aeruginosa from the healthy ocular surface in vivo. Healthy corneas of Black Swiss mice were inoculated with 10⁷ or 10⁹ CFU of invasive (PAO1) or cytotoxic (6206) P. aeruginosa. Viable counts were performed on tear fluid collected at time points ranging from 3 to 14 h postinoculation. Healthy ocular surfaces cleared both P. aeruginosa strains efficiently, even when 10⁹ CFU was used: e.g., <0.01% of the original inoculum was recoverable after 3 h. Preexposure of eyes to bacteria did not enhance clearance. Clearance of strain 6206 (low protease producer), but not strain PAO1 (high protease producer), was delayed in SP-D gene-targeted (SP-D^−/−) knockout mice. A protease mutant of PAO1 (PAO1 lasA lasB aprA) was cleared more efficiently than wild-type PAO1, but this difference was negligible in SP-D^−/− mice, which were less able to clear the protease mutant. Experiments to study mechanisms for these differences revealed that purified elastase could degrade tear fluid SP-D in vivo. Together, these data show that SP-D can contribute to the clearance of P. aeruginosa from the healthy ocular surface and that proteases can compromise that clearance. The data also suggest that SP-D degradation in vivo is a mechanism by which P. aeruginosa proteases could contribute to virulence.Pseudomonas aeruginosa, an opportunistic pathogen, is a leading cause of bacterial keratitis. While normal, healthy human corneas remain resistant to infection, contact lens wear or corneal injury/surgery can enable susceptibility (5, 15, 26). The mechanisms by which these factors predispose to infection are not yet well understood.A murine scarification model has been used exclusively to study the pathogenesis of P. aeruginosa corneal infection (3, 9, 30). That model involves scratching the cornea with a sterile needle prior to adding bacteria, which enables bacteria to directly access the exposed stroma. The resulting disease resembles P. aeruginosa infection in people. More recently, we used a healing model of murine corneal infection to show that 6 h after scratching, the mouse cornea remains susceptible to infection, but by 12 h, it regains resistance to infection despite loss of barrier function to fluorescein staining (18). These injury models that enable P. aeruginosa to infect the cornea have led to a wealth of information about how infection develops and resulting pathology. Yet, the mechanisms by which the normal ocular surface remains healthy under normal circumstances have not been explored in vivo. This cannot be studied using a scratch model. The corneas'' ability to resist disease despite constant daily exposure to potential pathogens is remarkable, and learning about the mechanisms involved could help us to develop new therapies for disease of the eye and possibly other sites.Results from the 12-hour healing situation suggested that defense systems other than barrier function can protect the ocular surface against infection. These defenses could involve biochemical factors constitutively expressed or upregulated in response to injury or bacterial exposure. Candidate factors could include defensin or other antimicrobial peptides, secretory immunoglobulin A, and mucin glycoproteins (11, 14, 23).In this study we focused on surfactant protein D (SP-D), which we have previously shown is present at the ocular surface, is upregulated by P. aeruginosa or its antigens, and can protect corneal epithelial cells against invasion (27, 28). Others have shown that SP-D-deficient mice lose their capacity to recover from P. aeruginosa keratitis when the eye is made susceptible using an injury model (25). Here our aim was to explore the role of SP-D in protecting the healthy eye against bacterial colonization. Thus, we developed a novel null-infection model in which the cornea is not damaged prior to inoculation with P. aeruginosa. The objective was to allow bacteria to interact with the healthy ocular surface (intact cornea) to enable us to study host factors that normally protect the eye from developing infection when it is not susceptible and the potential role that bacterial factors might play in compromising those defenses. This new model was then used to test the hypothesis that SP-D contributes to the clearance of P. aeruginosa from the healthy ocular surface and to explore the role of P. aeruginosa proteases in promoting ocular colonization in vivo.     

Isolation and Characterization of Amyloid-Related Serum Protein SAA as a Low Molecular Weight Protein

With direct immunoprecipitation or gel filtration under dissociating conditions, amyloid-related serum protein SAA has been isolated as a low molecular weight protein from the serum of two patients with rheumatoid arthritis but without known amyloidosis. The isolated protein SAA showed antigenic identity and an amino acid composition that was similar, but not identical, with isolated fibril protein AA. Molecular weight estimations suggest that protein SAA is approximately 50% larger than protein AA and has a molecular weight of 14,000-15,000 daltons. Preliminary results indicate that protein SAA from a patient with amyloidosis has a similar small molecular weight subunit.     

抗TfR单链抗体-AP融合蛋白的构建、表达与鉴定

目的构建、表达和鉴定抗转铁蛋白受体(TfR)单链抗体———碱性磷酸酶(AP)融合蛋白。方法用SfiⅠ和NotⅠ分别酶切抗转铁蛋白受体scFv表达质粒pUC19/119,获得scFv基因,直接亚克隆到表达载体pDAP2中,在TG1菌中表达scFvAP融合蛋白,SDSPAGE分析scFvAP的分子量,酶免疫测定鉴定其抗体和酶活性。结果scFvAP融合蛋白表达载体经scFv特异性引物PCR扩增,扩增产物经琼脂糖凝胶电泳可见约700bp条带,符合scFv基因理论值大小,IPTG诱导产物经SDSPAGE分析可见约为75ku的蛋白条带,符合scFvAP融合蛋白分子量的理论值大小,直接细胞ELISA测定证明表达产物scFvAP融合蛋白具有结合人TfR和AP活性的双重功效。结论抗人scFvAP融合蛋白表达载体构建成功,表达产物具有结合人TfR和AP活性的双重功效,为其临床检测应用奠定了基础。     

TMEM107 Is a Critical Regulator of Ciliary Protein Composition and Is Mutated in Orofaciodigital Syndrome

The proximate causes of multiple human genetic syndromes (ciliopathies) are disruptions in the formation or function of the cilium, an organelle required for a multitude of developmental processes. We previously identified Tmem107 as a critical regulator of cilia formation and embryonic organ development in the mouse. Here, we describe a patient with a mutation in TMEM107 that developed atypical Orofaciodigital syndrome (OFD), and show that the OFD patient shares several morphological features with the Tmem107 mutant mouse including polydactyly and reduced numbers of ciliated cells. We show that TMEM107 appears to function within cilia to regulate protein content, as key ciliary proteins do not localize normally in cilia derived from the Tmem107 mouse mutant and the human patient. These data indicate that TMEM107 plays a key, conserved role in regulating ciliary protein composition, and is a novel candidate for ciliopathies of unknown etiology.     

Expression and Purification of the Major Outer Membrane Protein of Chlamydia Trachomatis in Prokaryotic Cell

Chlamydiatrachomatis(C.trachomatis)isanobligate intracellularbacterialpathogen.Ocularinfectionwith C.trachomatisserovarsA,B,BaandCleadstotracho ma,aleadingcauseofpreventableblindnessinmanyde velopingcountries[1].Urogenital tractinfectionwithC. trachomat…     

人巨细胞病毒融合蛋白pp150/MDBP二级结构及其抗原表位预测

用Garnier-Robson和Chou-Fasman方案预测人巨细胞病毒融合蛋白pp150/MDBP的α-螺旋、β-折叠及转角的二级结构,用Kyte-Doolittle及Hopp-Woods亲水性方案预测其B细胞表位。研究结果表明,pp150/MDBP融合蛋白可能含有较少的α-螺旋,但可能含有较多的β-折叠及转角;B细胞识别的表位可能在7~56氨基酸残基或附近以及137~192残基或附近。     

人PD-L1Ig融合蛋白的表达及其生物学活性的研究

为获取人PD L1Ig融合蛋白 ,研究其对T细胞的调节效应 ,采用PCR法从pMD18 T/PD L1中扩增出人PD L1基因的胞外段序列 ,从人脾脏细胞中扩增出人IgG1Fc恒定区基因 ,两者拼接后插入逆转录病毒载体pEGZ Term ,用脂质体法与两个辅助病毒载体共转染 2 93T包装细胞 ,用含病毒颗粒的培养上清反复感染L92 9细胞 ,Zeocin筛选能稳定分泌人PD L1Ig蛋白的基因转染细胞并亚克隆之 ,经无血清培养 ,收集的上清用Dotblot检测、ELISA定量后 ,浓缩并经ProteinG柱纯化 ,再经Westernblot鉴定。以FACS分析纯化蛋白对活化T细胞表达的PD 1结合能力 ,以体外T细胞活化体系观察其对T细胞表型的调节作用。结果表明 ,成功地构建了表达人PD L1Ig蛋白的重组逆转录病毒载体 ;获得的L92 9/PD L1Ig细胞能稳定分泌人PD L1Ig蛋白 ;该蛋白与PD 1受体具有良好的结合能力 ,能抑制T细胞的进一步活化。     

Antibodies to 60-Kilodalton Heat Shock Protein and Outer Membrane Protein 2 of Chlamydia pneumoniae in Patients with Coronary Heart Disease

Evidence linking Chlamydia pneumoniae infection to atherosclerosis and to atherothrombotic events has recently emerged. A primary candidate implicated in these pathogenetic events is the 60-kDa chlamydial heat shock protein (HSP60). Another putative candidate to activate a potential proinflammatory mechanism is the chlamydial outer membrane protein 2 (OMP2). We have generated both HSP60 and OMP2 recombinant antigens in a nondenatured form and shown that (i) the two antigens were highly immunogenic in mice and (ii) murine antisera thus generated recognized the native C. pneumoniae proteins. We measured by enzyme linked immunosorbent assay (ELISA) and immunoblot assay antibody titers to the recombinant antigens in samples from 219 patients with coronary heart disease (CHD), 179 patients with unstable angina (UA), 40 patients with acute myocardial infarction (AMI), and 100 age-, sex-, and risk factor-matched healthy controls. We also examined whether anti-HSP60 and/or anti-OMP2 antibodies correlated with anti-C. pneumoniae antibodies assessed by a commercial microimmunofluorescence (MIF) assay. Immunoglobulin G (IgG), but neither IgA nor IgM, antibodies against the two recombinant proteins were detected by ELISA. In particular, anti-HSP60 antibodies were detected in >99% of CHD patients versus 0% of the controls, whereas the proportions of anti-OMP2 positive subjects were >70 and 27%, respectively. Nonetheless, among CHD patients, similar frequencies of positive subjects and titers of anti-HSP60 or anti-OMP2 antibodies were present in UA and AMI subjects. The anti-OMP2, but not the anti-HSP60, antibodies showed high specificity. Consistently, high serological correlation was observed between IgG MIF titers and IgG ELISA reactivity to OMP2 but not to HSP60. Overall, the results of this study demonstrate a strong correlation between CHD and anti-HSP60 IgG levels, as measured by our in-house ELISA. They also suggest that recombinant OMP2 ELISA, because of its high specificity and strong correlation with MIF assay, could be a candidate diagnostic marker for C. pneumoniae infection, which would be of potential usefulness for its specificity and nonsubjective nature.     

Modified C-Reactive Protein Is Expressed by Stroke Neovessels and Is a Potent Activator of Angiogenesis In Vitro

Native C-reactive protein (nCRP) is a pentameric oligo-protein and an acute phase reactant whose serum expression is increased in patients with inflammatory disease. We have identified by immunohistochemistry, significant expression of a tissue-binding insoluble modified version or monomeric form of CRP (mCRP) associated with angiogenic microvessels in peri-infarcted regions of patients studied with acute ischaemic stroke. mCRP, but not nCRP was expressed in the cytoplasm and nucleus of damaged neurons. mCRP co-localized with CD105, a marker of angiogenesis in regions of revascularisation. In vitro investigations demonstrated that mCRP was preferentially expressed in human brain microvessel endothelial cells following oxygen-glucose deprivation and mCRP (but not column purified nCRP) associated with the endothelial cell surface, and was angiogenic to vascular endothelial cells, stimulating migration and tube formation in matrigel more strongly than fibroblast growth factor-2. The mechanism of signal transduction was not through the CD16 receptor. Western blotting showed that mCRP stimulated phosphorylation of the key down-stream mitogenic signalling protein ERK1/2. Pharmacological inhibition of ERK1/2 phosphorylation blocked the angiogenic effects of mCRP. We propose that mCRP may contribute to the neovascularization process and because of its abundant presence, be important in modulating angiogenesis in both acute stroke and later during neuro-recovery.