BACKGROUND & AIMS: We studied changes in glutamate receptors, expression of immediate early genes, and AP-1 DNA binding activity in the brains of phenobarbital (PB)-dependent and -withdrawn rats to investigate the possible involvement of activation of glutamate receptors in PB withdrawal syndrome. PB-dependent rats were prepared by feeding drug-admixed food for 5 weeks. Autoradiographic analysis showed that binding of [3H(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imin e (MK-801), an antagonist of N-methyl-D-aspartic acid (NMDA) receptors, increased significantly in the cerebral cortices of PB-dependent and 24-h-withdrawn rats. However, [3H]MK-801 binding in the hippocampus and [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [3H]kainic acid binding in the hippocampus and cerebral cortex were essentially unchanged in both groups. PB withdrawal seizures were followed by increased expression of c-fos mRNA in the hippocampus and cerebral cortex and of c-jun mRNA in the cerebral cortex. The induction of c-fos and c-jun mRNA was suppressed by administration of MK-801. Furthermore, PB withdrawal enhanced AP-1 DNA binding activity in the brain. The present findings suggest functional enhancement of glutamatergic neurotransmission during the development of PB withdrawal syndrome.

背景与目标： 我们研究了苯巴比妥 (PB) 依赖性和退缩大鼠大脑中谷氨酸受体的变化，即刻早期基因的表达以及AP-1的DNA结合活性，以研究谷氨酸受体激活在PB戒断综合征中的可能参与。通过喂养混合药物的食物5周制备PB依赖性大鼠。放射自显影分析显示，N-甲基-d-天冬氨酸 (NMDA) 受体拮抗剂 [3H(+)-5-甲基-10,11-二氢-5H-二苯并 [a，D] cyclohepten-5，10-敏e (MK-801) 的结合，PB依赖性和24h撤回大鼠的大脑皮层显着增加。然而，[3h] MK-801在海马和 [3H]6-氰基-7-硝基喹喔啉-2结合，海马和大脑皮层中的3-二酮 (CNQX) 和 [3H] 海藻酸结合在两组中基本上没有变化。铅戒断发作后，海马和大脑皮层中c-fos mRNA的表达增加，大脑皮层中c-6月mRNA的表达增加。诱导c-MK-801可抑制fos和c-6月mRNA。此外，铅戒断增强了大脑中的AP-1 DNA结合活性。目前的发现表明，在铅戒断综合征的发展过程中，谷氨酸能神经传递的功能增强。

BACKGROUND & AIMS: :The extracellular calcium-sensing receptor (CaR) is usually associated with systemic Ca(2+) homeostasis, but the CaR is also expressed in many other tissues, including pancreatic islets of Langerhans. In the present study, we have used human islets and an insulin-secreting cell line (MIN6) to investigate the effects of CaR activation using the calcimimetic R-568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca(2+). CaR activation initiated a marked but transient insulin secretory response from both human islets and MIN6 cells at a sub-stimulatory concentration of glucose, and further enhanced glucose-induced insulin secretion. CaR-induced insulin secretion was reduced by inhibitors of phospholipase C or calcium-calmodulin-dependent kinases, but not by a protein kinase C inhibitor. CaR activation was also associated with an activation of p42/44 mitogen-activated protein kinases (MAPK), and CaR-induced insulin secretion was reduced by an inhibitor of p42/44 MAPK activation. We suggest that the beta-cell CaR is activated by divalent cations co-released with insulin, and that this may be an important mechanism of intra-islet communication between beta-cells.

背景与目标： : 细胞外钙感应受体 (CaR) 通常与全身Ca(2) 稳态有关，但CaR也在许多其他组织中表达，包括朗格汉斯的胰岛。在本研究中，我们使用人类胰岛和胰岛素分泌细胞系 (MIN6) 来研究使用钙拟R-568 (一种在细胞外Ca(2) 的生理浓度下激活CaR的CaR激动剂) 激活CaR的作用。CaR激活在葡萄糖的亚刺激浓度下启动了人类胰岛和MIN6细胞的显着但短暂的胰岛素分泌反应，并进一步增强了葡萄糖诱导的胰岛素分泌。磷脂酶C或钙-钙调蛋白依赖性激酶的抑制剂可减少CaR诱导的胰岛素分泌，但蛋白激酶C抑制剂不能减少。CaR激活也与p42/44丝裂原活化蛋白激酶 (MAPK) 的激活有关，并且CaR诱导的胰岛素分泌被p42/44 MAPK激活的抑制剂减少。我们建议 β 细胞CaR被与胰岛素共同释放的二价阳离子激活，这可能是 β 细胞之间胰岛内通讯的重要机制。

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： : 生物体组织的生物矿化依赖于优先使矿物质成核并控制其生长的蛋白质。此过程通常称为 “模板”，但该术语已成为通用术语，表示各种拟议的矿物-有机相互作用，包括化学和结构亲和力。在这里，我们提出了一种使用弹性蛋白和纤连蛋白纤维的自组装网络的方法，类似于细胞外基质。当在带负电荷的磺化聚苯乙烯表面上诱导时，这些蛋白质形成约10-mum间距的纤维网络，在它们之间留下无序蛋白质的开放区域。我们介绍了一种基于原子力显微镜的技术，用于在碳酸钙矿化之前和过程中测量结构化和杂乱无章的蛋白质的弹性模量。在矿化的早期阶段，矿物引起的蛋白质纤维的增稠和硬化得到了清楚的证明，早在离散的矿物晶体足够大以通过原子力显微镜成像之前。碳酸钙选择性地使蛋白质纤维变硬，而不会影响它们之间的区域，从而强调了矿物质与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进行的后期观察表明，Ca沿蛋白质纤维集中，并且晶体优先在纤维交叉上形成。我们证明，有组织的蛋白质与非结构化的蛋白质可以仅在纳米之间组装并在相同的环境中进行探测，在这种环境中，矿化被证明需要由细胞外基质的原纤维形成所施加的结构组织。

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的质膜蛋白和高效液相色谱纯化的晶状体的主要内在蛋白 (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/脂质比率成正比。在高比率下，这些颗粒像透镜纤维中的MIP一样结晶成四方阵列。由纯化的MIP或去污剂溶解的蛋白质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的电压下降至最大值的0.49 +/- 0.08。两态玻尔兹曼分布很好地拟合了电导对电压的依赖性。大于30 mV的电压阶跃引起欧姆电流阶跃，随后缓慢 (秒) 双指数下降。振幅和时间常数取决于幅度，而不取决于电压的符号。从100 mV到电压小于30 mV的阶跃导致通道以毫秒时间常数呈指数打开。对电压阶跃后首次闭合的潜伏期进行分析，得出的时间常数与多通道动力学几乎相同。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，其幅度为380和160 pS，以及三个额外的子状态。多通道和单通道数据表明该通道具有两个在动力学上重要的开放状态。通道具有轻微的阴离子选择性。通道的性质在pH 7.4至5.8或pca7至2之间没有明显变化。我们建议具有这些特性的通道可以有助于维持镜片的透明度和流体平衡。

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： : 检测了介导溶血磷脂酸 (LPA) 刺激的PKD(2) 激活的信号通路，以及PKD(2) 在调节LPA诱导的白介素8 (IL-8) 分泌中的潜在作用。未转化的人结肠上皮NCM460细胞。用LPA处理血清剥夺的NCM460细胞导致PKD(2) 的快速和惊人的激活，如通过体外激酶测定和激活环 (Ser706/710) 和自磷酸化位点 (Ser876) 测量的。通过与选择性PKC抑制剂gf-i预孵育消除LPA诱导的PKD(2) 激活，并以剂量依赖性方式Ro-31-8220。这些抑制剂对PKD(2) 活性没有任何直接抑制作用。通过NF-κ b-DNA结合，NF-κ b驱动的荧光素酶报告活性和ikappaba磷酸化来测量，LPA诱导了IL-8产生的显着增加并刺激了NF-κ b活化。利用靶向不同PKD(2) 序列的小干扰rna沉默PKD(2) 基因显著降低LPA刺激的NF-κ b启动子活性和IL-8产生。PKD(2) 激活是LPA生物学作用中的一个新的早期事件，并通过NF-κ b依赖性途径介导NCM460细胞中LPA刺激的IL-8分泌。我们的结果首次证明了PKD家族成员参与了上皮细胞产生IL-8 (一种有效的促炎趋化因子)。

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，它们的表达可以通过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，这些顺式作用调节基序之一是核因子X2 (NF-X2) 结合的X2-box。在这里，我们介绍了编码NF-X2的全长cDNA克隆隔离和表征。通过表达cDNA克隆分离该cDNA克隆，并编码人c 6月蛋白，该蛋白与c-Fos一起形成异二聚体激活蛋白1转录复合物。尽管b细胞中不存在c-Fos/c-6月异二聚体，但它们在II类非表达细胞中形成并结合X2-box。因此，c-Fos/c-6月异二聚体可能有助于抑制DRA基因表达。

BACKGROUND & AIMS: :The hypoxic environment in tumor is reported to play an important role in pancreatic cancer progression. The interaction between stromal and cancer cells also contributes to the malignant behavior of pancreatic cancer. In the present study, we investigated whether hypoxic stimulation affects stromal as well as pancreatic cancer cells. Our findings demonstrated that hypoxia remarkably elevated the HIF-1alpha expression in both pancreatic cancer (PK8) and fibroblast cells (MRC5). Hypoxic stimulation accelerated the invasive activity of PK8 cells, and invasiveness was thus further accelerated when the hypoxic PK8 cells were cultured with conditioned medium prepared from hypoxic MRC5 cells (hypoxic conditioned medium). MMP-2, MMP-7, MT1-MMP and c-Met expressions were increased in PK8 cells under hypoxia. Hypoxic stimulation also increased the hepatocyte growth factor (HGF) secretion from MRC5 cells, which led to an elevation of c-Met phosphorylation in PK8 cells. Conversely, the elevated cancer invasion, MMP activity and c-Met phosphorylation of PK8 cells were reduced by the removal of HGF from hypoxic conditioned medium. In immunohistochemical study, the HIF-1alpha expression was observed in surrounding stromal as well as pancreatic cancer cells, thus indicating hypoxia exists in both of cancer and stromal cells. Moreover, the stromal HGF expression was found to significantly correlate with not only the stromal HIF-1alpha expression but also the c-Met expression in cancer cells. These results indicate that the hypoxic environment within stromal as well as cancer cells activates the HGF/c-Met system, thereby contributing to the aggressive invasive features of pancreatic cancer.

背景与目标： : 据报道，肿瘤中的低氧环境在胰腺癌的进展中起重要作用。基质细胞和癌细胞之间的相互作用也有助于胰腺癌的恶性行为。在本研究中，我们调查了缺氧刺激是否会影响基质细胞以及胰腺癌细胞。我们的发现表明，缺氧显着提高了胰腺癌 (PK8) 和成纤维细胞 (MRC5) 的HIF-1alpha表达。低氧刺激加速了PK8细胞的侵袭活性，因此，当用低氧MRC5细胞制备的条件培养基 (低氧条件培养基) 培养低氧PK8细胞时，其侵袭能力进一步加快。缺氧条件下PK8细胞MMP-2、MMP-7、MT1-MMP和c-Met表达增加。低氧刺激还增加了MRC5细胞的肝细胞生长因子 (HGF) 分泌，导致PK8细胞中c-Met磷酸化升高。相反，通过从低氧条件培养基中去除HGF，可以降低PK8细胞的癌症侵袭，MMP活性和c-Met磷酸化水平。在免疫组织化学研究中，在周围基质以及胰腺癌细胞中观察到HIF-1alpha表达，因此表明癌症和基质细胞中都存在缺氧。此外，发现基质HGF表达不仅与癌细胞中的基质HIF-1alpha表达显着相关，而且与c-Met表达显着相关。这些结果表明，基质和癌细胞内的缺氧环境激活了HGF/c-Met系统，从而有助于胰腺癌的侵袭性特征。

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：

BACKGROUND & AIMS: :Related outer membrane proteins, termed secretins, participate in the secretion of macromolecules across the outer membrane of many Gram-negative bacteria. In the pullulanase-secretion system, PulS, an outer membrane-associated lipoprotein, is required both for the integrity and the proper outer membrane localization of the PulD secretin. Here we show that the PulS-binding site is located within the C-terminal 65 residues of PulD. Addition of this domain to the filamentous phage secretin, pIV, or to the unrelated maltose-binding protein rendered both proteins dependent on PulS for stability. A chimeric protein composed of bacteriophage f1 pIV and the C-terminal domain of PuID required properly localized PulS to support phage assembly. An in vivo complex formed between the pIV-PulD65 chimera and PulS was detected by co-immunoprecipitation and by affinity chromatography.

背景与目标： : 相关的外膜蛋白，称为分泌素，参与许多革兰氏阴性细菌外膜的大分子分泌。在支链淀粉酶分泌系统中，PulS (一种与外膜相关的脂蛋白) 对于PulD分泌素的完整性和适当的外膜定位都是必需的。在这里，我们显示了PulS结合位点位于PulD的C末端65个残基内。将该结构域添加到丝状噬菌体分泌素pIV或不相关的麦芽糖结合蛋白中，这两种蛋白质都依赖于PulS的稳定性。由噬菌体f1 pIV和PuID的C端结构域组成的嵌合蛋白需要适当定位的PulS来支持噬菌体组装。通过共免疫沉淀和亲和色谱法检测在pIV-PulD65嵌合体和PulS之间形成的体内复合物。

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) 是各种蛋白质的结构折叠和构象完整性维持所必需的，包括与细胞信号传导相关的几种。利用HSP90抑制剂17-allylamino-17-demethoxygeldanamycin (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是否可以靶向多发性骨髓瘤 (MM) 患者，我们首先通过免疫荧光和流式细胞仪分析研究了HSP90在骨髓瘤细胞系 (U266) 和原发性骨髓瘤细胞中的表达。在证明骨髓瘤细胞中HSP90表达后，通过免疫过氧化物酶染色分析了32 MM患者的档案样本。所有患者的骨髓瘤细胞在所有样品中均显示出HSP90的强细胞质表达，并且55% 还显示出同时的核免疫阳性。与未处理的对照细胞相比，用17AAG处理U266和原代MM细胞可显着增加细胞凋亡。对与17-aag孵育的MM细胞中抗凋亡BCL2家族蛋白和akt的分析表明，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17AAG和硼替佐米的组合治疗显示出对U266细胞系的协同凋亡作用。这些数据表明，靶向抑制HSP90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。

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： : 在先前对胰腺癌中差异表达基因的大规模筛选中，我们鉴定了一个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 (含KH结构域的蛋白质在癌症中过表达) 的基因分配到7p11.5号染色体上。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 5 '-UTR、1740 bp ORF和2168 bp 3'-UTR。科威特石油公司富含非盟的3 '-非翻译区包含八个AUUUA和四个auuuuuua重申的图案。推导的具有580个氨基酸的koc蛋白具有约65,000 (65 K) 的相对分子质量 (Mr)。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KH结构域已被证明参与RNA合成和代谢的调节，因此我们推测koc可能通过干扰转录和或转录后过程而在肿瘤细胞增殖的调节中发挥作用。然而，koc在人类肿瘤细胞中的确切作用尚不清楚，尚待阐明。

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8天大的结核分枝杆菌H37Ra培养滤液中分离出六种分子量不同的分泌蛋白 (15、26、30、41、55和70 kDa)。进一步检查了这些蛋白质在用总培养滤液蛋白免疫的小鼠中诱导细胞介导的 (T细胞增殖测定) 和体液免疫反应 (ELISA) 的能力。在六种蛋白质中，三种蛋白质显示出良好的反应性。然而，30 kDa抗原的活性最大。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，并发现其具有剂量依赖性。由该蛋白诱导的T细胞反应向T辅助 (Th1) 细胞倾斜，这是由interleukin-2 (IL-2) 和 γ-干扰素 (IFN-γ) 的明显分泌决定的。还评估了30 kDa蛋白的保护活性，并将其与卡介苗 (BCG) 疫苗进行了比较，该疫苗在用强毒力结核分枝杆菌H37Rv攻击的小鼠中进行了比较。30 kDa抗原在死亡率的基础上提供的保护程度，以及在用结核分枝杆菌H37Rv的LD50攻击30天后从不同器官 (肺，肝，脾) 恢复的c.f.u.的显着降低与BCG接种的动物相比更高。然而，该抗原诱导的免疫程度随时间而降低 (预防接种后8周和12周受到攻击时)，但仍与BCG相当。这些发现表明，结核分枝杆菌的30 kDa分泌蛋白是关键的免疫保护性抗原，并且可能是开发针对结核病的替代亚单位疫苗的合适候选者。

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：

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： : 来自鲨鱼 (软骨细胞) 的髓磷脂碱性蛋白 (MBP) 由比人类MBP更简单的电荷异构体混合物组成。应用于CM-52纤维素阳离子交换柱的总量的约3分之2在柱的未结合部分中回收; 剩余的3分之1结合到柱上并作为单个OD280峰洗脱。这种结合的材料没有播种人或牛MBP常见的电荷微异质性模式。未结合的级分由高分子量蛋白质 (55-60 kDa) 组成，该高分子量蛋白质构成该蛋白质级分的大部分，而低分子量蛋白质 (约18 kDa)。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) 增加约三倍，而Arg含量仅为牛或人类来源的18.5 kDa变体的约25%。该蛋白质中存在羟脯氨酸 (1.2残基/100) 是值得注意的，其鉴定是通过在两个不同系统中的氨基酸分析和通过质谱法实现的。在柱前衍生法中，羟脯氨酸在2.7分钟洗脱; 在柱后衍生法中，它在12.2分钟洗脱。通过快速原子轰击质谱分析完成了羟脯氨酸的鉴定。正在研究羟脯氨酸对该蛋白二级结构的影响。通过免疫学方法证实了这种高分子量蛋白在其一级结构中含有MBP序列。(摘要截短于250字)

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，是d-e-a-D (Asp-Glu-Ala-Asp) 盒蛋白家族的成员。它具有ATP依赖性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。分离并测序了编码一种称为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至49倍。当GBP与Gu/RH-II相互作用时，发生Gu/RH-II的蛋白水解裂解; Gu/RH-II的氨基末端部分对于这种蛋白水解至关重要。