• 【自组装蛋白纤维上的模板化生物矿化。】 复制标题 收藏 收藏
    DOI:10.1073/pnas.0602952103 复制DOI
    作者列表:Subburaman K,Pernodet N,Kwak SY,DiMasi E,Ge S,Zaitsev V,Ba X,Yang NL,Rafailovich M
    BACKGROUND & AIMS: :Biological mineralization of tissues in living organisms relies on proteins that preferentially nucleate minerals and control their growth. This process is often referred to as "templating," but this term has become generic, denoting various proposed mineral-organic interactions including both chemical and structural affinities. Here, we present an approach using self-assembled networks of elastin and fibronectin fibers, similar to the extracellular matrix. When induced onto negatively charged sulfonated polystyrene surfaces, these proteins form fiber networks of approximately 10-mum spacing, leaving open regions of disorganized protein between them. We introduce an atomic force microscopy-based technique to measure the elastic modulus of both structured and disorganized protein before and during calcium carbonate mineralization. Mineral-induced thickening and stiffening of the protein fibers during early stages of mineralization is clearly demonstrated, well before discrete mineral crystals are large enough to image by atomic force microscopy. Calcium carbonate stiffens the protein fibers selectively without affecting the regions between them, emphasizing interactions between the mineral and the organized protein fibers. Late-stage observations by optical microscopy and secondary ion mass spectroscopy reveal that Ca is concentrated along the protein fibers and that crystals form preferentially on the fiber crossings. We demonstrate that organized versus unstructured proteins can be assembled mere nanometers apart and probed in identical environments, where mineralization is proved to require the structural organization imposed by fibrillogenesis of the extracellular matrix.
    背景与目标: :生物体内组织的生物矿化依赖蛋白质优先使矿物质成核并控制其生长。该过程通常被称为“模板化”,但该术语已变得通用,表示各种提议的矿物-有机相互作用,包括化学亲和力和结构亲和力。在这里,我们提出一种使用弹性蛋白和纤连蛋白纤维自组装网络的方法,类似于细胞外基质。当被诱导到带负电荷的磺化聚苯乙烯表面上时,这些蛋白质形成大约10微米间距的纤维网络,在它们之间留下了杂乱的蛋白质的开放区域。我们引入基于原子力显微镜的技术来测量碳酸钙矿化前后的结构化和无组织蛋白的弹性模量。在矿化的早期阶段,很明显地证明了矿物质诱导的蛋白质纤维的增厚和变硬,早在离散的矿物晶体足够大以至于无法通过原子力显微镜成像时。碳酸钙选择性地使蛋白质纤维变硬而不影响它们之间的区域,强调了矿物质和有组织的蛋白质纤维之间的相互作用。通过光学显微镜和二次离子质谱的后期观察显示,Ca沿着蛋白质纤维集中,并且晶体优先在纤维交叉处形成。我们证明有组织的与非结构化的蛋白质可以仅相隔纳米而组装在一起,并在相同的环境中进行探测,在该环境中,矿化被证明需要细胞外基质的原纤维形成所强加的结构组织。
  • 【由晶状体纤维膜的主要内在蛋白重建的通道特性。】 复制标题 收藏 收藏
    DOI:10.1085/jgp.96.3.631 复制DOI
    作者列表:Ehring GR,Zampighi G,Horwitz J,Bok D,Hall JE
    BACKGROUND & AIMS: :Detergent-solubilized plasma membrane protein of either adult bovine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramembrane particles in the vesicles was proportional to the protein/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays as does MIP in lens fibers. Channels induced by either purified MIP or detergent-solubilized protein had essentially identical properties. The conductance of multichannel membranes was maximal near 0 mV and decreased to 0.49 +/- 0.08 of the maximum value at voltages greater than 80 mV. The dependence of the conductance on voltage was well fit by a two-state Boltzmann distribution. Voltage steps greater than 30 mV elicited an ohmic current step followed by a slow (seconds) biexponential decrease. The amplitudes and time constants depended on the magnitude but not the sign of the voltage. Steps from 100 mV to voltages less than 30 mV caused the channels to open exponentially with a millisecond time constant. Analysis of latency to first closure after a voltage step gave nearly the same time constants as multichannel kinetics. Single-channel conductance is proportional to salt concentration from 0.1 to 1.0 M in KCl. In 0.1M KCl, the channel had two preferred conductance states with amplitudes of 380 and 160 pS, as well as three additional substates. Multi- and single-channel data suggest that the channel has two kinetically important open states. The channel is slightly anion selective. The properties of the channel do not vary appreciably from pH 7.4 to 5.8 or from pCa 7 to 2. We propose that a channel with these properties could contribute to maintenance of lens transparency and fluid balance.
    背景与目标: :成年牛或小牛晶状体的去污剂溶解质膜蛋白和高效液相色谱纯化的晶状体主要内在蛋白(MIP)重构为单层囊泡和平面脂质双层。冷冻断裂研究表明,囊泡中膜内颗粒的密度与蛋白质/脂质比成正比。在高比例下,这些颗粒与透镜纤维中的MIP一样结晶为四边形阵列。纯化的MIP或去污剂溶解的蛋白诱导的通道具有基本相同的特性。多通道膜的电导在0 mV附近最大,并且在大于80 mV的电压下降低到最大值的0.49 /-0.08。电导对电压的依赖性通过两态玻耳兹曼分布很好地拟合。大于30 mV的电压阶跃引起欧姆电流阶跃,然后缓慢(秒)双指数下降。幅度和时间常数取决于幅度,而不取决于电压的符号。从100 mV到低于30 mV的电压阶跃导致通道以毫秒的时间常数呈指数方式打开。电压阶跃后首次闭合的等待时间的分析给出了与多通道动力学几乎相同的时间常数。单通道电导与KCl中0.1至1.0 M的盐浓度成正比。在0.1M KCl中,通道具有两个优选的电导状态,其振幅分别为380和160 pS,以及三个其他子状态。多通道和单通道数据表明该通道具有两个动力学上重要的开放状态。该通道对阴离子具有轻微的选择性。通道的特性在pH 7.4至5.8或pCa 7至2范围内变化不大。我们建议具有这些特性的通道可有助于维持镜片的透明度和流体平衡。
  • 【蛋白激酶D2通过NF-κB介导未转化的人结肠上皮细胞中溶血磷脂酸诱导的白介素8的产生。】 复制标题 收藏 收藏
    DOI:10.1152/ajpcell.00308.2006 复制DOI
    作者列表:Chiu TT,Leung WY,Moyer MP,Strieter RM,Rozengurt E
    BACKGROUND & AIMS: :The signaling pathways mediating lysophosphatidic acid (LPA)-stimulated PKD(2) activation and the potential contribution of PKD(2) in regulating LPA-induced interleukin 8 (IL-8) secretion in nontransformed, human colonic epithelial NCM460 cells were examined. Treatment of serum-deprived NCM460 cells with LPA led to a rapid and striking activation of PKD(2), as measured by in vitro kinase assay and phosphorylation at the activation loop (Ser706/710) and autophosphorylation site (Ser876). PKD(2) activation induced by LPA was abrogated by preincubation with selective PKC inhibitors GF-I and Ro-31-8220 in a dose-dependent manner. These inhibitors did not have any direct inhibitory effect on PKD(2) activity. LPA induced a striking increase in IL-8 production and stimulated NF-kappaB activation, as measured by NF-kappaB-DNA binding, NF-kappaB-driven luciferase reporter activity, and IkappaBalpha phosphorylation. PKD(2) gene silencing utilizing small interfering RNAs targeting distinct PKD(2) sequences dramatically reduced LPA-stimulated NF-kappaB promoter activity and IL-8 production. PKD(2) activation is a novel early event in the biological action of LPA and mediates LPA-stimulated IL-8 secretion in NCM460 cells through a NF-kappaB-dependent pathway. Our results demonstrate, for the first time, the involvement of a member of the PKD family in the production of IL-8, a potent proinflammatory chemokine, by epithelial cells.
    背景与目标: :审查了介导溶血磷脂酸(LPA)刺激的PKD(2)激活和PKD(2)在调节LPA诱导的非转化型人结肠上皮NCM460细胞中LPA诱导的白介素8(IL-8)分泌中的潜在作用。用体外LPA处理血清缺乏的NCM460细胞会导致PKD(2)迅速而惊人的活化,这是通过体外激酶测定和活化环(Ser706 / 710)和自磷酸化位点(Ser876)的磷酸化来测量的。通过与选择性PKC抑制剂GF-1和Ro-31-8220呈剂量依赖性方式进行预孵育,可以消除LPA诱导的PKD(2)激活。这些抑制剂对PKD(2)活性没有任何直接的抑制作用。如通过NF-κB-DNA结合,NF-κB驱动的萤光素酶报道分子活性和IkappaBalpha磷酸化所测量,LPA诱导IL-8产量显着增加并刺激NF-κB活化。 PKD(2)基因沉默利用针对不同的PKD(2)序列的小干扰RNA,大大降低了LPA刺激的NF-κB启动子活性和IL-8的产生。 PKD(2)激活是LPA的生物学作用中的一个新的早期事件,并通过NF-κB依赖性途径介导LCM刺激NCM460细胞中IL-8的分泌。我们的结果首次证明,上皮细胞参与了PKD家族成员参与IL-8(一种有效的促炎趋化因子)的生产。
  • 【与DRA X2-box结合的NF-X2是激活蛋白1。c-Jun的表达克隆。】 复制标题 收藏 收藏
    DOI: 复制DOI
    作者列表:Andersson G,Peterlin BM
    BACKGROUND & AIMS: :Human class II MHC Ag are a family of cell surface glycoproteins. Their constitutive expression is limited to B lymphocytes and thymic epithelial cells. In many other cells their expression can be induced by IFN-gamma. Conserved upstream promoter sequences regulate this tissue-specific expression of class II genes. In the DRA promoter, one of these cis-acting regulatory motifs is the X2-box to which nuclear factor X2 (NF-X2) binds. Here, we present the isolation and characterization of the full-length cDNA clone encoding NF-X2. This cDNA clone was isolated by expression cDNA cloning, and encodes the human c-Jun protein, which together with c-Fos forms the heterodimeric activator protein-1 transcription complex. Whereas c-Fos/c-Jun heterodimers do not exist in B cells, they form and bind to the X2-box in class II nonexpressing cells. Thus, c-Fos/c-Jun heterodimers might contribute to the repression of DRA gene expression.
    背景与目标: :人类II类MHC Ag是细胞表面糖蛋白家族。它们的组成型表达仅限于B淋巴细胞和胸腺上皮细胞。在许多其他细胞中,它们的表达可以被IFN-γ诱导。保守的上游启动子序列调节II类基因的这种组织特异性表达。在DRA启动子中,这些顺式作用调控基元之一是X2-box,其与核因子X2(NF-X2)结合。在这里,我们介绍了编码NF-X2的全长cDNA克隆的分离和表征。该cDNA克隆通过表达cDNA克隆进行分离,并编码人c-Jun蛋白,该蛋白与c-Fos一起形成异二聚激活蛋白-1转录复合体。尽管B细胞中不存在c-Fos / c-Jun异二聚体,但它们在II类非表达细胞中形成并与X2-box结合。因此,c-Fos / c-Jun异二聚体可能有助于抑制DRA基因表达。
  • 【Exogean:用于注释真核基因组DNA中蛋白质编码基因的框架。】 复制标题 收藏 收藏
    DOI:10.1186/gb-2006-7-s1-s7 复制DOI
    作者列表:Djebali S,Delaplace F,Roest Crollius H
    BACKGROUND & AIMS: BACKGROUND:Accurate and automatic gene identification in eukaryotic genomic DNA is more than ever of crucial importance to efficiently exploit the large volume of assembled genome sequences available to the community. Automatic methods have always been considered less reliable than human expertise. This is illustrated in the EGASP project, where reference annotations against which all automatic methods are measured are generated by human annotators and experimentally verified. We hypothesized that replicating the accuracy of human annotators in an automatic method could be achieved by formalizing the rules and decisions that they use, in a mathematical formalism. RESULTS:We have developed Exogean, a flexible framework based on directed acyclic colored multigraphs (DACMs) that can represent biological objects (for example, mRNA, ESTs, protein alignments, exons) and relationships between them. Graphs are analyzed to process the information according to rules that replicate those used by human annotators. Simple individual starting objects given as input to Exogean are thus combined and synthesized into complex objects such as protein coding transcripts. CONCLUSION:We show here, in the context of the EGASP project, that Exogean is currently the method that best reproduces protein coding gene annotations from human experts, in terms of identifying at least one exact coding sequence per gene. We discuss current limitations of the method and several avenues for improvement.
    背景与目标: 背景:在真核生物基因组DNA中进行准确,自动的基因鉴定比以往任何时候都更重要,它对于有效利用社区可利用的大量组装基因组序列至关重要。一直以来,人们一直认为自动方法的可靠性不如人类专业知识。这在EGASP项目中得到了说明,其中由人工注释者生成了针对其测量所有自动方法的参考注释,并对其进行了实验验证。我们假设可以通过以数学形式主义形式化规则和决策来实现以自动方式复制人类注释器的准确性。
    结果:我们开发了Exogean,这是一个基于有向无环彩色多图(DACM)的灵活框架,可以表示生物对象(例如,mRNA,EST,蛋白质比对,外显子)及其之间的关系。根据复制人类注释者使用的规则的规则,对图形进行分析以处理信息。因此,作为输入到Exogean的简单单个起始对象将被合并并合成为复杂的对象,例如蛋白质编码转录本。
    结论:在EGASP项目的背景下,我们证明了Exogean目前是从人类专家中复制蛋白质编码基因注释的最佳方法,因为每个基因至少可以识别一个确切的编码序列。我们讨论了该方法的当前局限性和改进的几种途径。
  • 【多发性骨髓瘤中热休克蛋白90(HSP90)的表达和HSP90抑制剂(17-AAG)的作用分析。】 复制标题 收藏 收藏
    DOI:10.1080/10428190500472123 复制DOI
    作者列表:Duus J,Bahar HI,Venkataraman G,Ozpuyan F,Izban KF,Al-Masri H,Maududi T,Toor A,Alkan S
    BACKGROUND & AIMS: :Heat shock protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated an antitumor effect in solid tumors. To test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analysed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55% also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins and akt in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL, MCL-1 and akt. Furthermore, although a low concentration of bortezomib resulted in no cell death, a combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.
    背景与目标: :热休克蛋白90(HSP90)是结构折叠和维持各种蛋白质(包括与细胞信号相关的几种蛋白质)构象完整性的必需。利用HSP90抑制剂17-烯丙基氨基-17-去甲氧基格尔德霉素(17-AAG)的最新研究表明,在实体瘤中具有抗肿瘤作用。为了测试HSP90是否可以靶向于多发性骨髓瘤(MM)患者,我们首先通过免疫荧光和流式细胞术分析了骨髓瘤细胞系(U266)和原发性骨髓瘤细胞中HSP90的表达。在证明HSP90在骨髓瘤细胞中表达后,通过免疫过氧化物酶染色分析了32例MM患者的档案样本。在所有患者中,骨髓瘤细胞在所有样品中均表现出强烈的HSP90细胞质表达,并且55%的患者还表现出并发的核免疫阳性。与未处理的对照细胞相比,用17AAG处理U266细胞和原代MM细胞可导致凋亡明显增加。分析与17-AAG孵育的MM细胞中的抗凋亡BCL2家族蛋白和akt,表明BCL-2,BCL-XL,MCL-1和akt下调。此外,尽管低浓度的硼替佐米不会导致细胞死亡,但是17AAG和硼替佐米治疗的组合显示出对U266细胞系的协同凋亡作用。这些数据表明,针对HSP90的靶向抑制可能被证明是开发MM患者未来治疗选择的有效且创新的策略。
  • 【克隆一种在癌症中高度过表达的基因,该基因编码一种新型的含有KH域的蛋白质。】 复制标题 收藏 收藏
    DOI:10.1038/sj.onc.1201110 复制DOI
    作者列表:Müeller-Pillasch F,Lacher U,Wallrapp C,Micha A,Zimmerhackl F,Hameister H,Varga G,Friess H,Büchler M,Beger HG,Vila MR,Adler G,Gress TM
    BACKGROUND & AIMS: :In a previous large scale screen for differentially expressed genes in pancreatic cancer, we identified a gene highly overexpressed in cancer encoding a novel protein with four K-homologous (KH) domains. KH-domains are found in a subset of RNA-binding proteins, including pre-mRNA-binding (hnRNP) K protein and the fragile X mental retardation gene product (FMR1). By fluorescence in situ hybridization (FISH) the identified gene named koc (KH domain containing protein overexpressed in cancer) was assigned to chromosome 7p11.5. Two pseudogenes were localised on chromosome 6 and 11. The cloned koc cDNA has a 250 bp 5'-UTR, a 1740 bp ORF and a 2168 bp 3'-UTR. The AU-rich 3'-untranslated region of koc contains eight AUUUA and four AUUUUUA reiterated motifs. The deduced koc protein with 580 amino-acids has a relative molecular mass (Mr) of approximately 65,000 (65 K). The koc transcript is highly overexpressed in pancreatic cancer cell lines and in pancreatic cancer tissue as compared to both, normal pancreas and chronic pancreatitis tissue. High levels of expression were as well found in tissue samples of other human tumours. As the KH domain has been shown to be involved in the regulation of RNA synthesis and metabolism, we speculate that koc may assume a role in the regulation of tumour cell proliferation by interfering with transcriptional and or posttranscriptional processes. However, the precise role of koc in human tumour cells is unknown and remains to be elucidated.
    背景与目标: :在先前针对胰腺癌中差异表达基因的大规模筛选中,我们鉴定了在癌症中高度过表达的基因,该基因编码具有四个K同源(KH)域的新型蛋白质。 KH结构域存在于RNA结合蛋白的子集中,包括前mRNA结合(hnRNP)K蛋白和脆弱的X智力低下基因产物(FMR1)。通过荧光原位杂交(FISH),将鉴定出的名为koc(在癌症中过表达的KH域蛋白)的基因分配给7p11.5染色体。两个假基因位于6号和11号染色体上。克隆的koc cDNA具有250 bp的5'-UTR,1740 bp的ORF和2168 bp的3'-UTR。富含AU的3'非翻译区域的koc包含八个AUUUA和四个AUUUUUA重复的基序。推导的具有580个氨基酸的koc蛋白的相对分子质量(Mr)约为65,000(65 K)。与正常胰腺和慢性胰腺炎组织相比,koc转录本在胰腺癌细胞系和胰腺癌组织中高度过表达。在其他人类肿瘤的组织样本中也发现了高水平的表达。由于已经显示出KH结构域参与RNA合成和代谢的调节,我们推测koc可能通过干扰转录和/或转录后过程而在调节肿瘤细胞增殖中发挥作用。但是,koc在人肿瘤细胞中的确切作用尚不清楚,尚待阐明。
  • 【结核分枝杆菌H37Ra 30 kDa分泌蛋白的免疫生物学特性。】 复制标题 收藏 收藏
    DOI:10.1016/s0264-410x(96)00230-7 复制DOI
    作者列表:Sinha RK,Verma I,Khuller GK
    BACKGROUND & AIMS: :Six different secretory proteins of molecular weights (15, 26, 30, 41, 55 and 70 kDa) were isolated from 8-day-old culture filtrate of Mycobacterium tuberculosis H37Ra using different column chromatography techniques. These proteins were further examined for their ability to induce cell mediated (T-cell proliferation assay) and humoral immune response (ELISA) in mice immunized with total culture filtrate proteins. Out of six proteins, three proteins showed good reactivity. However, the activity was at a maximum with 30 kDa antigen. The immune response induced by 30 kDa antigen emulsified in Freund's incomplete adjuvant (FIA) was investigated and was found to be dose dependent. The T-cell response induced by this protein was skewed towards T-helper (Th1) cells as determined by the pronounced secretion of interleukin-2 (IL-2) and gamma-interferon (IFN-gamma). The protective activity of the 30 kDa protein was also evaluated and compared with reference to Bacillus Calmette Guerin (BCG) vaccine in the mice challenged with virulent M. tuberculosis H37Rv. The degree of protection afforded by the 30 kDa antigen on the basis of mortality and the significant decrease in c.f.u.'s recovered from different organs (lung, liver, spleen) after 30 days of challenge with LD50 of M. tuberculosis H37Rv was significantly higher in comparison to BCG vaccinated animals. However, the degree of immunity induced by this antigen decreased with time (when challenged 8 and 12 weeks post-immunization) but it was still comparable with BCG. These findings suggest that 30 kDa secretory protein of M. tuberculosis is the key immunoprotective antigen and may be a suitable candidate for the development of an alternative subunit vaccine against tuberculosis.
    背景与目标: :使用不同的柱色谱技术从结核分枝杆菌H37Ra的8天龄培养滤液中分离出六个分子量分别为15、26、30、41、55和70 kDa的分泌蛋白。在用总培养滤液蛋白免疫的小鼠中,进一步检查了这些蛋白诱导细胞介导的能力(T细胞增殖测定)和体液免疫应答(ELISA)。在六种蛋白质中,三种蛋白质显示出良好的反应性。但是,对于30kDa抗原,活性最大。研究了在弗氏不完全佐剂(FIA)中乳化的30 kDa抗原诱导的免疫反应,发现该反应是剂量依赖性的。该蛋白诱导的T细胞反应偏向T辅助(Th1)细胞,这由白介素2(IL-2)和γ-干扰素(IFN-γ)的明显分泌确定。还评估了30 kDa蛋白的保护活性,并与卡介苗芽孢杆菌(BCG)疫苗进行了比较,比较了在有毒结核分枝杆菌H37Rv攻击的小鼠中的行为。 30 kDa抗原提供的保护程度基于死亡率以及结核分枝杆菌H37Rv的LD50攻击30天后从不同器官(肺,肝,脾)回收的cfu显着降低。与接种卡介苗的动物比较。但是,这种抗原诱导的免疫度随时间降低(在免疫后8周和12周激发时),但仍可与BCG相提并论。这些发现表明,结核分枝杆菌的30kDa分泌蛋白是关键的免疫保护性抗原,并且可能是开发替代性抗结核亚单位疫苗的合适候选者。
  • 【受体激活剂NFkappaB配体和骨保护素蛋白在人根尖囊肿和肉芽肿中的表达。】 复制标题 收藏 收藏
    DOI:10.1016/j.tripleo.2005.10.054 复制DOI
    作者列表:Menezes R,Bramante CM,da Silva Paiva KB,Letra A,Carneiro E,Fernando Zambuzzi W,Granjeiro JM
    BACKGROUND & AIMS: OBJECTIVE:The purpose of this study was to determine the expression of receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin (OPG) associated with bone destruction in periapical cysts and granulomas. STUDY DESIGN:Forty human dental chronic periapical lesions were collected after periapical surgery. The lesions collected were fixed in 10% buffered formalin and histologically processed. At least 2 sections of each specimen were stained with hematoxylin and eosin for microscopic diagnosis. After that, 10 human periapical granulomas and 10 cysts were selected for immunohistochemical analysis for RANKL, OPG, and CD68+. RESULTS:Polymorphonuclear neutrophils, macrophages, endothelial cells, and lymphocytes were stained for RANKL and OPG in both lesions. Epithelial cells were also stained for RANKL and OPG in periapical cysts. Quantitative analysis was conducted and the results were expressed as a ratio of the number of immunostained cells over the total number of cells in the field (n = 100). The ratio of RANKL+/total cells was higher than OPG+/total cells in periapical granulomas (0.553 +/- 0.153 and 0.483 +/- 0.189, respectively; P < .0012; paired t test) and in cysts (0.519 +/- 0.09 and 0.339 +/- 0.117, respectively; P < .0001; paired t test). The ratios of OPG+/total cells (P < .0001; paired t test) and RANKL+/total cells (P < .0322; paired t test) were greater in granulomas than in cysts. However, the ratio RANKL+/OPG+ in granulomas (1.336 +/- 0.723) and cysts (1.404 +/- 0.385) was not significantly different. The ratio of CD68+/total cells was significantly higher in granulomas (0.381 +/- 0.040) than in cysts (0.307 +/- 0.068) (P < .0001; unpaired t test with Welch correction). CONCLUSION:Taking into account the limitations of the experimental approach employed, our findings indicate the presence of RANKL and OPG in cysts and granulomas, strongly suggesting the involvement of these gene products in the development of periapical lesions.
    背景与目标: 目的:本研究旨在确定与根尖周囊肿和肉芽肿中的骨破坏有关的NFkappaB配体(RANKL)和骨保护素(OPG)受体激活剂的表达。
    研究设计:根尖周手术后收集了40例人类牙齿慢性根尖周病变。将收集的病灶固定在10%的福尔马林缓冲液中,并进行组织学处理。每个标本至少2个切片用苏木精和曙红染色以进行显微镜诊断。之后,选择10个人根尖肉芽肿和10个囊肿进行RANKL,OPG和CD68的免疫组织化学分析。
    结果:在两个病变中,多形核中性粒细胞,巨噬细胞,内皮细胞和淋巴细胞均进行了RANKL和OPG染色。还对上皮周囊肿中的上皮细胞进行了RANKL和OPG染色。进行定量分析,结果表示为免疫染色细胞数与现场细胞总数之比(n = 100)。根尖肉芽肿中RANKL /总细胞的比率高于OPG /总细胞的比率(分别为0.553 /-0.153和0.483 /-0.189; P <.0012;成对t检验)和囊肿中(0.519 /-0.09和0.339 / -分别为0.117; P <.0001;成对t检验)。肉芽肿中OPG /总细胞(P <.0001;成对t检验)与RANKL /总细胞(P <.0322;成对t检验)的比率大于囊肿。然而,肉芽肿(1.336 /-0.723)和囊肿(1.404 /-0.385)中的RANKL / OPG比率没有显着差异。肉芽肿中CD68 /总细胞的比率(0.381 /-0.040)显着高于囊肿(0.307 /-0.068)(P <.0001;采用Welch校正的未配对t检验)。
    结论:考虑到所用实验方法的局限性,我们的发现表明囊肿和肉芽肿中存在RANKL和OPG,强烈暗示这些基因产物参与了根尖周病变的发展。
  • 【鲨鱼脑中一种含有羟脯氨酸的蛋白质,与髓磷脂碱性蛋白质有关。】 复制标题 收藏 收藏
    DOI:10.1111/j.1471-4159.1990.tb04958.x 复制DOI
    作者列表:Wood DD,McLaurin J,Moscarello MA
    BACKGROUND & AIMS: :Myelin basic protein (MBP) from shark (Chondricthyes) consists of a simpler mixture of charge isomers than human MBP. About two-thirds of the total amount applied to a CM-52 cellulose cation-exchange column was recovered in the unbound fraction of the column; the remaining one-third bound to column and was eluted as a single OD280 peak. This bound material did not sow the usual pattern of charge microheterogeneity found with human or bovine MBP. The unbound fraction was composed of a high molecular weight protein (55-60 kDa), which constituted most of this protein fraction and a low molecular weight protein (approximately 18 kDa). The amino acid composition of our unbound fraction was similar to that reported earlier. The Glx (glutamic acid + glutamine) was increased about threefold whereas the Arg content was only about 25% of that of the 18.5 kDa variant of bovine or human origin. The presence of hydroxyproline (1.2 residues/100) in this protein was noteworthy, identification of which was achieved by amino acid analysis in two different systems and by mass spectrometry. In the precolumn derivatization method, hydroxyproline eluted at 2.7 min; in the postcolumn derivatization method it eluted at 12.2 min. Identification of hydroxyproline was completed by fast atom bombardment-mass spectral analysis. The effect of hydroxyproline on the secondary structure of this protein is being studied. Verification that this high molecular weight protein contained MBP sequences within its primary structure was confirmed by immunological methods.(ABSTRACT TRUNCATED AT 250 WORDS)
    背景与目标: :鲨鱼(Chondricthyes)的髓磷脂碱性蛋白(MBP)由电荷异构体组成的混合物比人MBP更简单。应用于CM-52纤维素阳离子交换柱的总量的约三分之二是在该柱的未结合馏分中回收的;剩余的三分之一与色谱柱结合,并被洗脱为一个OD280峰。这种结合的材料并未播种人或牛MBP常见的电荷微异质性模式。未结合的部分由高分子量蛋白质(55-60 kDa)和低分子量蛋白质(约18 kDa)组成,其中高分子量蛋白质占该蛋白质部分的大部分。我们未结合部分的氨基酸组成与先前报道的相似。 Glx(谷氨酸谷氨酰胺)增加了约三倍,而Arg含量仅为牛或人来源的18.5 kDa变体的Arg含量的约25%。值得注意的是,该蛋白质中存在羟脯氨酸(1.2个残基/ 100个),通过在两个不同系统中进行氨基酸分析并通过质谱法进行鉴定。在柱前衍生化方法中,羟脯氨酸在2.7分钟洗脱;在柱后衍生化方法中,它在12.2分钟时洗脱。通过快速原子轰击质谱分析完成了羟脯氨酸的鉴定。羟脯氨酸对该蛋白二级结构的影响正在研究中。通过免疫学方法证实了这种高分子量蛋白质在其一级结构中包含MBP序列。(摘要截短为250字)
  • 【Gu / RH-II结合蛋白的克隆和鉴定。】 复制标题 收藏 收藏
    DOI:10.1006/bbrc.1997.6642 复制DOI
    作者列表:Valdez BC,Henning D,Perlaky L,Busch RK,Busch H
    BACKGROUND & AIMS: :Gu/RNA helicase II (Gu/RH-II) is the first reported mammalian nucleolar RNA helicase that is a member of the D-E-A-D (Asp-Glu-Ala-Asp) box family of proteins. It has an ATP-dependent RNA unwinding (helicase) activity and a separate RNA folding activity (introduction of intramolecular secondary structure into single-stranded RNA). To determine which proteins may bind to Gu/RH-II, a yeast two-hybrid system was used. A cDNA which encoded a protein, called Gu/RH-II binding protein or GBP, was isolated and sequenced. The GBP protein is localized to the nucleus in speckled or diffuse nucleoplasmic patterns. The GBP mRNA level is highest in testis, 9- to 49-fold greater than other tissues. When GBP interacts with Gu/RH-II, proteolytic cleavage of Gu/RH-II occurs; the amino-terminal portion of Gu/RH-II is critical for this proteolysis.
    背景与目标: :Gu / RNA解旋酶II(Gu / RH-II)是第一个报道的哺乳动物核仁RNA解旋酶,它是D-E-A-D(Asp-Glu-Ala-Asp)盒蛋白家族的成员。它具有ATP依赖的RNA解旋(解旋酶)活性和单独的RNA折叠活性(将分子内二级结构引入单链RNA)。为了确定哪些蛋白质可以与Gu / RH-II结合,使用了酵母双杂交系统。分离并测序了编码称为Gu / RH-II结合蛋白或GBP的蛋白质的cDNA。 GBP蛋白以斑点或弥散的核质模式定位于细胞核。 GBP mRNA水平在睾丸中最高,比其他组织高9到49倍。当GBP与Gu / RH-II相互作用时,会发生Gu / RH-II的蛋白水解切割。 Gu / RH-II的氨基末端部分对于这种蛋白水解至关重要。
  • 【差异磷酸肌醇与G蛋白门控K通道的成分结合。】 复制标题 收藏 收藏
    DOI:10.1007/s00232-006-0014-5 复制DOI
    作者列表:Thomas AM,Brown SG,Leaney JL,Tinker A
    BACKGROUND & AIMS: :The regulation of ion channels and transporters by anionic phospholipids is currently very topical. G protein-gated K(+) channels from the Kir3.0 family are involved in slowing the heart rate, generating late inhibitory postsynaptic potentials and controlling hormone release from neuroendocrine cells. There is considerable functional precedent for the control of these channels by phosphatidylinositol 4,5-bisphosphate. In this study, we used a biochemical assay to investigate the lipid binding properties of Kir3.0 channel domains. We reveal a differential binding affinity to a range of phosphoinositides between the C termini of the Kir3.0 isoforms. Furthermore, the N terminus in addition to the C terminus of Kir3.4 is necessary to observe binding and is decreased by the mutations R72A, K195A and R196A but not K194A. Protein kinase C phosphorylation of the Kir3.1 C-terminal fusion protein decreases anionic phospholipid binding. The differential binding affinity has functional consequences as the inhibition of homomeric Kir3.1, occurring after M3 receptor activation, recovers over minutes while homomeric Kir3.2 does not.
    背景与目标: :阴离子磷脂对离子通道和转运蛋白的调节目前非常热门。 Kir3.0家族的G蛋白门控的K()通道参与减慢心率,产生晚期抑制的突触后电位并控制神经内分泌细胞的激素释放。磷脂酰肌醇4,5-双磷酸酯对这些通道的控制有相当大的功能先例。在这项研究中,我们使用生化分析来研究Kir3.0通道域的脂质结合特性。我们揭示了对Kir3.0亚型的C末端之间的一系列磷酸肌醇的不同结合亲和力。此外,除了观察Kir3.4的C末端外,N末端对于观察结合也是必需的,并且由于突变R72A,K195A和R196A而不是K194A而降低。 Kir3.1 C端融合蛋白的蛋白激酶C磷酸化减少了阴离子磷脂的结合。差异结合亲和力具有功能性后果,因为抑制M3受体激活后发生的同源Kir3.1抑制会在数分钟内恢复,而同源Kir3.2不会恢复。
  • 【黄色荧光蛋白变体YFP-H148Q / I152L的碘化钠转运蛋白活性的基于细胞的成像。】 复制标题 收藏 收藏
    DOI:10.1152/ajpcell.00291.2006 复制DOI
    作者列表:Rhoden KJ,Cianchetta S,Stivani V,Portulano C,Galietta LJ,Romeo G
    BACKGROUND & AIMS: :The sodium iodide symporter (NIS) mediates iodide (I(-)) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I(-)-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I(-) induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I(-) (35 muM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na(+) dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I(-) influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I(-) influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I(-) also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I(-) uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I(-) may be a useful tool by which to study the pathophysiology and pharmacology of NIS.
    背景与目标: :碘化钠共转运蛋白(NIS)介导碘化物(I(-))在甲状腺和其他组织中的运输,并作为治疗靶标和核成像报告者越来越重要。目前,使用放射性示踪剂和电生理技术测量了NIS的体外活性。我们报告了使用I(-)敏感和遗传可编码的黄色荧光蛋白(YFP)变体YFP-H148Q / I152L对NIS活性进行新型活细胞成像测定的发展报告。在稳定表达YFP-H148Q / I152L的FRTL-5甲状腺细胞中,I(-)诱导了细胞荧光的快速和可逆下降,其特征在于1)对细胞外I(-)(35 muM)的高度亲和力,2)NIS抑制剂的抑制作用高氯酸盐,3)细胞外Na()依赖性和4)TSH依赖性,表明荧光变化是由于I(-)通过NIS流入所致。 FRTL-5细胞群中的单个细胞在NIS介导的I(-)流入速率中表现出3.5倍的变化,说明了YFP-H148Q / I152L可用于检测NIS活性中的细胞间差异。 I(-)还在表达NIS的COS-7细胞中引起高氯酸盐敏感的YFP-H148Q / I152L荧光下降,但在缺乏NIS的细胞中不引起高氯酸盐敏感性下降。这些结果表明,YFP-H148Q / I152L是基因转移后甲状腺细胞和非甲状腺细胞中NIS介导的I(-)摄取的灵敏生物传感器,并表明细胞I(-)的荧光检测可能是一种有用的工具,研究NIS的病理生理学和药理学。
  • 【蛋白质与基质配体的共沉淀:可缩放的蛋白质分离。】 复制标题 收藏 收藏
    DOI:10.1002/(sici)1099-1352(199634/12)9:5/6<433::aid-j 复制DOI
    作者列表:Matulis D,Lovrien R,Richardson TI
    BACKGROUND & AIMS: Matrix ligands are agents for isolating proteins out of dilute crudes by coprecipitating proteins. The ligands have a strong anion sulfonate head which initiates binding to proteins having a positive net charge, ZH+ approximately 5-20. Initial binding tightens protein conformation and starts to squeeze water from conformationally motile proteins. The tails are stackable hydrophobic organic groups, azoaromatic dyes which draw protein-ligand complexes together. Proteins coprecipitate as guests, in the ligand host matrix. In addition to stacking, ligand tails displace water because of their bulk, and lower the average dielectric constant near charged groups, which reinforces the electrostatic component of binding. Matrix ligands protect proteins, scavenge them from dilute crudes (0.01-0.1 per cent protein), and densify coprecipitates. Detergent ions in low concentrations, 10(-4)-10(-5) M also sometimes serve as coprecipitating agents, entangling their tails but probably not stacking. Divalent metal ions, Zn++, sometimes are useful auxiliary agents. Preparative scaleability from crudes is demonstrated starting from 100-200 g of raw peanuts and raw pineapple to coprecipitate a lectin and bromelain enzyme respectively in 1-2 h with 80-90 per cent activity yields. Ligands are released from coprecipitates by shifting the pH and trapping the ligands with exchange resins. Protein conformation tightening in solution is seen by viscosity measurements.

    背景与目标: 基质配体是通过共沉淀蛋白质从稀原油中分离蛋白质的试剂。配体具有很强的阴离子磺酸根头,可与具有正净电荷(ZH约为5-20)的蛋白质结合。初始结合会拉紧蛋白质构象,并开始从构象运动的蛋白质中挤出水。尾部是可堆叠的疏水性有机基团,偶氮芳香族染料,可将蛋白质-配体复合物吸引到一起。蛋白质在配体宿主基质中作为客人共沉淀。除堆积之外,配体尾部由于其体积大而会置换水,并降低带电基团附近的平均介电常数,从而增强了结合的静电成分。基质配体可以保护蛋白质,从稀原油中清除蛋白质(蛋白质含量为0.01-0.1%),并浓缩共沉淀物。低浓度10(-4)-10(-5)M的去污剂离子有时还充当共沉淀剂,缠住它们的尾巴,但可能不会堆积。二价金属离子Zn有时是有用的辅助剂。从100-200 g的生花生和菠萝开始,可在1-2小时内分别沉淀出凝集素和菠萝蛋白酶,从而产生80-90%的活性收率,证明了从原油中可制备的可缩放性。通过改变pH值并用交换树脂捕获配体,从共沉淀物中释放出配体。通过粘度测量可以看出溶液中的蛋白质构象变紧。

  • 【植物73 kDa过氧化物酶体膜蛋白(PMP73)与分子伴侣具有免疫相关性。】 复制标题 收藏 收藏
    DOI: 复制DOI
    作者列表:Corpas FJ,Trelease RN
    BACKGROUND & AIMS: We previously showed via electron microscopic immunocytochemistry that a 73 kDa polypeptide was an authentic peroxisomal membrane protein (PMP73) integrated exclusively into the boundary membrane of glyoxysomes in cucumber seedlings. In this paper we test the hypothesis that this PMP73 is a member of the heat-shock 70 protein (Hsp70) family by comparing amino acid sequences of cyanogen bromide (CNBr)-cleaved polypeptide fragments, immunoreactivities on protein blots, and microscopic immunofluorescence within suspension-cultured BY-2 tobacco cells. A sequence of eight amino acids (DAVGPEIQ) in PMP73 showed a high degree of similarity (up to 88%) with sequences in the same carboxy-terminal region of four plant Hsp70 proteins. IgGs affinity purified to PMP73 recognized on blots a membrane-bound Hsp72 (in pea cotyledon microsomes) and a cucumber PMP61, the latter shown by CNBr cleavage to be a distinct, but immunorelated polypeptide to PMP73. Conversely, IgGs specific for tomato Hsc70 (C-terminal half) recognized cucumber PMP73, and IgGs specific for cucumber DnaJ homologue (entire protein) recognized cucumber PMP61. In BY-2 cells, cucumber PMP73-specific IgGs localized only to peroxisomes. Antibodies raised against portions of tomato Hsc70 also localized to the BY-2 peroxisomes (as well as to cytosolic proteins). Collectively, the data show that authentic cucumber PMPs73 and 61 are immunorelated to each another, and that both exhibit selective immunoreactivity to IgGs from two classes of molecular chaperones, namely Hsp70 proteins and plant DnaJ homologues. They appear to be unique membrane-bound chaperones that likely function as part of the peroxisomal protein translocation machinery.

    背景与目标: 我们先前通过电子显微镜免疫细胞化学表明,一个73 kDa的多肽是一种真正的过氧化物酶体膜蛋白(PMP73),仅整合到黄瓜幼苗中乙醛酸体的边界膜中。在本文中,我们通过比较溴化氰(CNBr)裂解的多肽片段的氨基酸序列,蛋白质印迹上的免疫反应性以及悬浮液中的微观免疫荧光,来验证这一PMP73是热休克70蛋白(Hsp70)家族成员的假设。 -培养的BY-2烟草细胞。 PMP73中的八个氨基酸序列(DAVGPEIQ)与四个植物Hsp70蛋白的相同羧基末端区域中的序列具有高度相似性(最高88%)。纯化至PMP73的IgG亲和力可在膜结合的Hsp72(在豌豆子叶微粒体中)和黄瓜PMP61上印迹,后者通过CNBr裂解显示是与PMP73不同但与免疫相关的多肽。相反,对番茄Hsc70特异的IgG(C端一半)识别黄瓜PMP73,对黄瓜DnaJ同源物(整个蛋白)特异的IgG识别黄瓜PMP61。在BY-2细胞中,黄瓜PMP73特异性IgG仅定位于过氧化物酶体。针对番茄Hsc70部分产生的抗体也定位于BY-2过氧化物酶体(以及胞质蛋白)。总体而言,数据表明,真实的黄瓜PMPs73和61彼此免疫相关,并且都显示出对来自两类分子伴侣(即Hsp70蛋白和植物DnaJ同源物)的IgG的选择性免疫反应性。它们似乎是独特的膜结合伴侣蛋白,可能是过氧化物酶体蛋白易位机制的一部分。

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