BACKGROUND & AIMS: Recent evidence suggests that Chlamydia trachomatis can persist in the female upper genital tract in an unculturable state. Since unsuspected C. trachomatis infection has been associated with adverse in-vitro fertilization (IVF) outcome we sought to detect further evidence of C. trachomatis in the genital tracts of women undergoing IVF. The prevalence and distribution of antibodies to the major structural proteins of C. trachomatis in paired follicular fluid and sera of women undergoing IVF were examined. Sera and follicular fluid samples from 149 women were assayed for immunoglobulin (Ig)G and IgA antibodies to two C. trachomatis antigens, the major outer membrane protein (MOMP) and a recombinant lipopolysaccharide (rLPS) fragment. Additionally, the expression of human 60 kDa heat shock protein (hsp 60) in follicular fluid was determined. All cervical and follicular fluid samples were negative for C. trachomatis by polymerase chain reaction, ligase chain reaction and DNA probe. Sera from 60% of the subjects were positive for antichlamydial rLPS IgG; 36% were positive for anti-MOMP IgG. Similarly, rLPS-directed and MOMP-directed IgA were detected in sera of 34 and 14% of the subjects respectively. IgG antibodies to MOMP and rLPS were detected in 42 and 41% of the follicular fluid examined respectively. Anti-MOMP IgA was identified in 8.7% of the follicular fluid while 27.5% were positive for anti-rLPS IgA. Human hsp 60 expression was documented in 11.6% of the follicular fluid tested. IgA antibodies to both MOMP (P = 0.03) and rLPS (P = 0.02) in follicular fluid were associated with a failure to become pregnant after embryo transfer. IgG antibodies in sera and follicular fluid and IgA antibodies in sera were unrelated to IVF outcome. Similarly only anti-MOMP IgA (P = 0.02) and anti-rLPS IgA (P = 0.04) in follicular fluid were correlated with human hsp 60 expression in follicular fluid. The unique association between IgA antibodies to two chlamydial antigens in follicular fluid and both hsp 60 expression and IVF failure provides further support for the possibility that a persistent upper genital tract chlamydial infection contributes to IVF failure in some women.

背景与目标： 最近的证据表明，沙眼衣原体可以在女性上生殖道中以不可培养的状态持续存在。由于未经怀疑的沙眼衣原体感染与不良的体外受精 (IVF) 结果有关，因此我们试图在接受IVF的妇女的生殖道中进一步发现沙眼衣原体的证据。检查了成对的卵泡液和接受IVF的妇女血清中沙眼衣原体主要结构蛋白的抗体的患病率和分布。对来自149名妇女的血清和卵泡液样品进行了免疫球蛋白 (Ig)G和IgA抗体的检测，该抗体针对两种沙眼衣原体抗原，主要外膜蛋白 (MOMP) 和重组脂多糖 (rLPS) 片段。此外，测定了人60 kDa热休克蛋白 (hsp 60) 在卵泡液中的表达。通过聚合酶链反应，连接酶链反应和DNA探针，所有宫颈和卵泡液样品均阴性沙眼衣原体。来自60% 名受试者的血清中抗衣原体rLPS IgG阳性; 36% 抗MOMP IgG阳性。同样，分别在34和14% 名受试者的血清中检测到rLPS导向和MOMP导向的IgA。分别在42和41% 的卵泡液中检测到针对MOMP和rLPS的IgG抗体。在卵泡液8.7% 中鉴定出抗MOMP IgA，而27.5% 对抗rLPS呈阳性igA。人类hsp 60的表达在测试的卵泡液11.6% 中被记录。卵泡液中MOMP (P = 0.03) 和rlp (P = 0.02) 的IgA抗体与胚胎移植后未能怀孕有关。血清和卵泡液中的IgG抗体和血清中的IgA抗体与IVF结果无关。同样，只有卵泡液中的抗MOMP IgA (P = 0.02) 和抗rLPS IgA (P = 0.04) 与人hsp 60在卵泡液中的表达相关。针对两种衣原体抗原的IgA抗体与hsp 60表达和IVF失败为某些女性持续的上生殖道衣原体感染导致IVF失败的可能性提供了进一步的支持。

BACKGROUND & AIMS: The attachment protein or G protein of the A2 strain of human respiratory syncytial virus (RSV) was digested with trypsin and the resultant peptides separated by reverse-phase high-performance liquid chromatography (HPLC). One tryptic peptide produced a mass by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) corresponding to residues 152-187 with the four Cys residues of the ectodomain (residues 173, 176, 182, and 186) in disulfide linkage and absence of glycosylation. Sub-digestion of this tryptic peptide with pepsin and thermolysin produced peptides consistent with disulfide bonds between Cys173 and Cys186 and between Cys176 and Cys182. Analysis of ions produced by post-source decay of a peptic peptide during MALDI-TOF-MS revealed fragmentation of peptide bonds with minimal fission of an inter-chain disulfide bond. Ions produced by this unprecedented MALDI-induced post-source fragmentation corroborated the existence of the disulfide arrangement deduced from mass analysis of proteolysis products. These findings indicate that the ectodomain of the G protein has a non-glycosylated subdomain containing a "cystine noose."

背景与目标： 用胰蛋白酶消化人呼吸道合胞病毒 (RSV) 的A2菌株的附着蛋白或g蛋白，并通过反相高效液相色谱 (HPLC) 分离所得肽。一种胰蛋白酶肽通过基质辅助激光解吸/电离 (MALDI) 飞行时间 (TOF) 质谱 (MS) 产生了与胞外域的四个Cys残基152-187的残基 (残基173，176，182，和186) 在二硫键连接和不存在糖基化。用胃蛋白酶和嗜热菌素对这种胰蛋白酶进行亚消化，产生的肽与Cys173和Cys186之间以及Cys176和cys182之间的二硫键一致。对maldi-tof-MS期间消化肽的源后衰变产生的离子的分析表明，肽键断裂，链间二硫键的裂变最小。这种前所未有的MALDI诱导的后源断裂产生的离子证实了从蛋白水解产物的质量分析得出的二硫键排列的存在。这些发现表明g蛋白的外结构域具有包含 “胱氨酸套索” 的非糖基化亚域。

BACKGROUND & AIMS: :Etanercept is a recombinant human soluble tumor necrosis factor (TNF-alpha) receptor fusion protein that inhibits TNF-alpha, a major mediator in the pathogenesis of graft-versus-host disease (GVHD). The purpose of our study was to evaluate the safety and efficacy of etanercept therapy in 21 patients with steroid-refractory acute GVHD (aGVHD) (n = 13) and chronic GVHD (cGVHD) (n = 8). Etanercept 25 mg was given subcutaneously twice weekly for 4 weeks followed by 25 mg weekly for 4 weeks. At the time of initiation of etanercept, 14 patients had skin, 13 had gastro-intestinal, 5 had liver, 5 had pulmonary, and 4 had oral involvement. Twelve patients (57%) completed 12 doses of therapy. Overall, 11 of 21 patients (52%) responded to the treatment with etanercept, including 6 patients (46%) with aGVHD [n = 4 complete response (CR), n = 2 partial response (PR)] and 5 patients (62%) with cGVHD (n = 1 CR, n = 4 PR). Clinical responses were most commonly seen in patients with refractory gut aGVHD with 55% of the patients having a CR and 9% having a PR. CMV reactivation occurred in 48% of patients, bacterial infections in 14% of patients, and fungal infections in 19% of patients. Fourteen patients (67%) were alive after a median follow-up of 429 days (range 71-1007 days) since initiation of etanercept. Seven patients died, 3 of infections, 2 of refractory aGVHD, and 2 of disease progression. In conclusion, our preliminary data indicate that etanercept is well tolerated and can induce a high response rate in patients with steroid-refractory aGVHD and cGVHD, particularly in the setting of GI involvement.

背景与目标： : 依那西普是一种重组人可溶性肿瘤坏死因子 (TNF-α) 受体融合蛋白，可抑制TNF-α，TNF-α 是移植物抗宿主病 (GVHD) 发病机理中的主要介质。我们研究的目的是评估依那西普治疗21例类固醇难治性急性GVHD (aGVHD) (n = 13) 和慢性GVHD (cGVHD) (n = 8) 患者的安全性和有效性。依那西普25 mg，每周皮下注射两次，持续4周，然后每周注射25 mg，持续4周。在开始使用依那西普时，14例患者有皮肤，13例有胃肠道，5例有肝脏，5例有肺部，4例有口腔受累。12名患者 (57%) 完成12剂治疗。总体而言，21例患者中有11例 (52%) 对依那西普治疗有反应，其中6例 (46% 例) aGVHD [n = 4完全缓解 (CR)，n = 2部分缓解 (PR)] 和5例 (62%) cGVHD (n = 1 CR，n = 4 PR)。临床反应最常见于难治性肠道aGVHD患者，其中55% 患者具有CR，9% 患者具有PR。48% 患者发生CMV再激活，14% 患者发生细菌感染，19% 患者发生真菌感染。自依那西普开始以来，中位随访429天 (范围71-1007天) 后，有14名患者 (67%) 还活着。7例患者死亡，3例感染，2例难治性aGVHD，2例疾病进展。总之，我们的初步数据表明，依那西普具有良好的耐受性，并且可以在类固醇难治性aGVHD和cGVHD患者中诱导高反应率，尤其是在GI受累的情况下。

BACKGROUND & AIMS: :The prevalence of IgG antibodies against Mycobacterium leprae recombinant culture filtrate protein-10 (rCFP-10) was investigated in serum samples from 56 leprosy patients, 15 tuberculosis (TB) patients, 14 other skin-diseased patients and 20 healthy subjects. On classifying the patients into bacterial index (BI)-positive and BI-negative groups, the assay showed 83.3 % (15/18) sensitivity for detection of BI-positive leprosy patients. On the other hand, the sensitivity for detection of BI-negative patients was 18.4 % (7/38). None of the 15 TB patients and 14 other skin-diseased patients was positive; however, only one out of 20 healthy individuals was positive, indicating that antibody response to culture filtrate protein-10 (CFP-10) was highly specific (98.0 %; 48/49). Statistically, the performance of the CFP-10-based assay was found to be comparable (P>0.05) with that of an anti-phenolic glycolipid-I (PGL-I) antibody-detecting assay. Thus, M. leprae CFP-10 is potentially a specific antigen for measuring antibody response in BI-positive leprosy patients. Being a secreted antigen, CFP-10 may act as a marker for the viability of M. leprae inside the host, and hence its serological potential is worth exploring for application in monitoring the response of patients with BI-positive leprosy (a highly infectious form) during the course of chemotherapy. When comparing the bacteriological and serological results, an agreement of 82.1 % showed that seropositivity to M. leprae CFP-10 corresponded well with bacteriological criteria. Hence, CFP-10 seems to be a suitable antigen for classification of leprosy patients into BI-positive and BI-negative groups.

背景与目标： : 在56名麻风病患者，15名结核病 (TB) 患者，14名其他皮肤病患者和20名健康受试者的血清样本中，研究了针对麻风分枝杆菌重组培养滤液蛋白10 (rCFP-10) 的IgG抗体的患病率。在将患者分为细菌指数 (BI) 阳性和BI阴性组时，该测定显示了检测BI阳性麻风病患者的83.3% (15/18) 敏感性。另一方面，检测双阴性患者的灵敏度18.4% (7/38)。15个TB患者和14个其他皮肤疾病患者中没有一个是阳性的; 然而，20个健康个体中只有一个是阳性的，表明对培养滤液蛋白10 (CFP-10) 的抗体反应是高度特异性的 (98.0%; 48/49)。统计学上，发现CFP-10-based测定的性能与抗-酚糖脂-I (pgl-i) 抗体检测测定的性能相当 (P>0.05)。因此，麻风分枝杆菌CFP-10潜在地是用于测量双阳性麻风病患者的抗体应答的特异性抗原。作为一种分泌的抗原，CFP-10可以作为宿主内麻风支原体生存能力的标志物，因此其血清学潜力值得探索，用于监测双阳性麻风病 (一种高度感染形式) 患者在化疗过程中的反应。当比较细菌学和血清学结果时，82.1% 的一致性表明麻风分枝杆菌的血清阳性CFP-10与细菌学标准非常吻合。因此，CFP-10似乎是用于将麻风病患者分为双阳性和双阴性组的合适抗原。

BACKGROUND & AIMS: :Iminodisuccinate (IDS) epimerase catalyzes the epimerisation of R,R-, S,S- and R,S- iminodisuccinate, one step in the biodegradation of the chelating agent iminodisuccinate by Agrobacterium tumefaciens BY6. The enzyme is a member of the MmgE/PrpD protein family, a diverse and little characterized class of proteins of prokaryotic and eukaryotic origin. IDS epimerase does not show significant overall amino acid sequence similarity to any other protein of known three-dimensional structure. The crystal structure of this novel epimerase has been determined by multi-wavelength diffraction to 1.5 A resolution using selenomethionine-substituted enzyme. In the crystal, the enzyme forms a homo-dimer, and the subunit consists of two domains. The larger domain, not consecutive in sequence and comprising residues Met1-Lys266 and Leu400-Pro446, forms a novel all alpha-helical fold with a central six-helical bundle. The second, smaller domain folds into an alpha+beta domain, related in topology to chorismate mutase by a circular permutation. IDS epimerase is thus not related in three-dimensional structure to other known epimerases. The fold of the IDS epimerase is representative for the whole MmgE/PrpD family. The putative active site is located at the interface between the two domains of the subunit, and is characterized by a positively charged surface, consistent with the binding of a highly negatively charged substrate such as iminodisuccinate. Docking experiments suggest a two-base mechanism for the epimerisation reaction.

背景与目标： : 亚氨基二琥珀酸酯 (IDS) 差向异构化催化R，R-，S，S-和R，S-亚氨基二琥珀酸酯的差向异构化，这是根癌农杆菌对螯合剂亚氨基二琥珀酸酯生物降解的第一步。该酶是MmgE/PrpD蛋白家族的成员，MmgE/PrpD蛋白家族是原核和真核来源的多种多样且特征很少的蛋白质。IDS差向异构酶与已知三维结构的任何其他蛋白质没有显示出明显的总体氨基酸序列相似性。该新型差向异构酶的晶体结构已通过多波长衍射确定，以使用硒代蛋氨酸取代的酶1.5分辨率。在晶体中，酶形成同型二聚体，亚基由两个结构域组成。较大的结构域不是连续的并且包含Met1-Lys266和Leu400-Pro446的残基，形成具有中心六螺旋束的新型全 α-螺旋折叠。第二个较小的结构域折叠成 α β 结构域，在拓扑上通过环状排列与脉络酸突变酶有关。因此，IDS差向异构酶在三维结构上与其他已知的差向异构酶无关。IDS差向异构酶的折叠代表整个MmgE/PrpD家族。推定的活性位点位于亚基的两个结构域之间的界面处，其特征在于带正电的表面，与高度带负电的底物 (例如亚氨基二琥珀酸酯) 的结合一致。对接实验提出了向异构化反应的两基机制。

BACKGROUND & AIMS: BACKGROUND:This study was undertaken to obtain differentially expressed genes related to human glioma by cDNA microarray and the characterization of a novel full-length gene. METHODS:Total RNA was extracted form human glioma and normal brain tissue, and mRNA was used as a probe. The results of hybridization procedure were scanned with the computer system. The gene named 507E08 cone was subsequently analyzed by northern blot, bioinformatic approach, and protein expression. RESULTS:Fifteen differentially expressed genes were obtained from human glioma by hybridization and scanning for four times. Northern blot analysis confirmed that the 507E08 clone was low expressed in human brain tissue and over expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that the 507E08 clone was a novel full-length gene, which codes 203 amino acid of protein and is called human ribosomal protein 14.22 gene. The nucleotide sequence had been submitted to the GenBank with the accession number of AF329277. After expression in E. coli., protein yielded a major band of apparent molecular mass 22 kDa on an SDS-PAGE gel. CONCLUSIONS:cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human ribosomal protein 13.22 may be correlated with the development of human glioma.

背景与目标：

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。