• 【procaspase-3的低氧特异性细胞毒性与含有Pro564的HIF-1alpha的VHL介导的蛋白质破坏基序融合的机制。】 复制标题 收藏 收藏
    DOI:10.1016/j.febslet.2006.09.025 复制DOI
    作者列表:Harada H,Kizaka-Kondoh S,Hiraoka M
    BACKGROUND & AIMS: :Under normoxic conditions the alpha-subunit of hypoxia-inducible factor (HIF-1alpha) protein is targeted for degradation by the von Hippel-Lindau (VHL) tumor suppressor protein acting as an E3 ubiquitin ligase. Recently, we developed a hypoxia-targeting protein, TOP3, which consisted of procaspase-3 with the VHL-mediated protein destruction motif of HIF-1alpha. This design enables procaspase-3 to be regulated similarly with HIF-1alpha, being degraded under normoxia while stabilized under hypoxia. Furthermore, stabilized TOP3 was cleaved by the hypoxic stress-induced endogenous caspases and thus the procaspase-3 was converted to active caspase-3 specifically under hypoxic conditions. These data demonstrated that the VHL-mediated protein destruction motif of HIF-1alpha endowed procaspase-3 with hypoxia-specific cytotoxicity.
    背景与目标: 在常氧条件下,缺氧诱导因子(HIF-1alpha)蛋白的α亚基被von Hippel-Lindau(VHL)肿瘤抑制蛋白作为E3泛素连接酶降解。最近,我们开发了一种低氧靶向蛋白TOP3,该蛋白由具有HIF-1alpha的VHL介导的蛋白破坏基序的procaspase-3组成。此设计使procaspase-3与HIF-1alpha相似,在常氧下降解,而在低氧下稳定。此外,稳定化的TOP3被低氧应激诱导的内源性半胱天冬酶裂解,因此procaspase-3特别是在低氧条件下转化为活性caspase-3。这些数据表明,HIF-1α的VHL介导的蛋白质破坏基序赋予了procaspase-3具有缺氧特异性细胞毒性。
  • 【对沙眼衣原体膜成分的体液免疫应答和体外受精患者卵泡液中人60 kDa热休克蛋白的表达。】 复制标题 收藏 收藏
    DOI:10.1093/humrep/12.5.925 复制DOI
    作者列表:Neuer A,Lam KN,Tiller FW,Kiesel L,Witkin SS
    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无法培养的状态持续存在。由于未曾怀疑的沙眼衣原体感染与体外受精(IVF)不良结果有关,因此我们寻求在接受IVF的女性生殖道中发现沙眼衣原体的进一步证据。检查了在进行IVF的女性的配对卵泡液和血清中沙眼衣原体主要结构蛋白抗体的流行情况和分布。分析了来自149名妇女的血清和卵泡液样品中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)片段的免疫球蛋白(Ig)G和IgA抗体。另外,测定了人60kDa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,所有子宫颈和卵泡液样品均为沙眼衣原体阴性。 60%的受试者血清抗衣原体rLPS IgG阳性; 36%的抗MOMP IgG阳性。同样,分别在34%和14%的受试者血清中检测到了rLPS导向和MOMP导向的IgA。在分别检查的卵泡液中有42%和41%检测到针对MOMP和rLPS的IgG抗体。在8.7%的卵泡液中发现了抗MOMP IgA,而抗rLPS IgA的阳性率为27.5%。在测试的卵泡液中有11.6%记录了人类hsp 60的表达。卵泡液中针对MOMP(P = 0.03)和rLPS(P = 0.02)的IgA抗体与胚胎移植后无法怀孕有关。血清和卵泡液中的IgG抗体以及血清中的IgA抗体与IVF结果无关。同样,卵泡液中只有抗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。卵泡液中针对两种衣原体抗原的IgA抗体与hsp 60表达和IVF衰竭之间的独特联系为某些女性持续性上生殖道衣原体感染导致IVF衰竭的可能性提供了进一步的支持。

  • 【通过基质辅助激光解吸/电离飞行时间质谱测定人呼吸道合胞病毒附着(G)蛋白的二硫键排列。】 复制标题 收藏 收藏
    DOI:10.1002/pro.5560060619 复制DOI
    作者列表:Gorman JJ,Ferguson BL,Speelman D,Mills J
    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)A2株的附着蛋白或G蛋白,并通过反相高效液相色谱(HPLC)分离得到的肽。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)产生质量,对应于带有胞外域的四个Cys残基的残基152-187(残基173、176, 182和186)中的二硫键和不存在糖基化作用。用胃蛋白酶和嗜热菌蛋白酶亚消化该胰蛋白酶消化的肽产生的肽与Cys173和Cys186之间以及Cys176和Cys182之间的二硫键一致。对在MALDI-TOF-MS过程中由消化性肽的源后衰减产生的离子进行的分析显示,肽键断裂且链间二硫键的裂变最小。由这种前所未有的MALDI诱导的源后裂解产生的离子证实了从蛋白水解产物的质量分析推导出的二硫键结构的存在。这些发现表明,G蛋白的胞外域具有一个非糖基化的亚域,其中包含一个“胱氨酸套索”。

  • 【重组人可溶性肿瘤坏死因子受体融合蛋白作为同种异体造血干细胞移植后类固醇难治性移植物抗宿主病的治疗方法。】 复制标题 收藏 收藏
    DOI:10.1002/ajh.20752 复制DOI
    作者列表:Busca A,Locatelli F,Marmont F,Ceretto C,Falda M
    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: :Etanercept是一种重组人类可溶性肿瘤坏死因子(TNF-alpha)受体融合蛋白,可抑制TNF-alpha(一种在移植物抗宿主病(GVHD)发病机理中的主要介体)。我们的研究目的是评估依那西普治疗21例激素抵抗性急性GVHD(aGVHD)(n = 13)和慢性GVHD(cGVHD)(n = 8)患者的安全性和有效性。每周两次皮下给予Etanercept 25 mg,持续4周,然后每周25 mg,持续4周。依那西普开始治疗时,有14例皮肤,13例胃肠,5例肝,5例肺,4例经口受累。 12名患者(57%)完成了12剂治疗。总体上,21例患者中有11例(52%)对依那西普治疗有反应,其中6例(46%)患有aGVHD [n = 4完全缓解(CR),n = 2部分缓解(PR)]和5例(62 %)和cGVHD(n = 1 CR,n = 4 PR)。临床反应最常见于顽固性肠aGVHD患者,其中55%的患者为CR,9%的患者为PR。 CMV重新激活发生在48%的患者中,细菌感染发生在14%的患者中,而真菌感染发生在19%的患者中。自依那西普开始接受中位随访429天(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死亡,2例疾病进展。总之,我们的初步数据表明,依那西普耐受性好,在类固醇难治性aGVHD和cGVHD患者中可引起较高的应答率,特别是在胃肠道受累的情况下。
  • 【在麻风病人中检测抗麻风分枝杆菌培养滤液蛋白10的抗体。】 复制标题 收藏 收藏
    DOI:10.1099/jmm.0.46587-0 复制DOI
    作者列表:Parkash O,Kumar A,Nigam A,Girdhar BK
    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: :从56名麻风患者,15名结核病患者,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抗麻风分枝杆菌重组培养物滤液蛋白-10(rCFP-10)IgG抗体的患病率。在将患者分为细菌指数(BI)阳性和BI阴性组后,该测定显示出83.3%(15/18)的灵敏度可检测BI阳性麻风病患者。另一方面,检测BI阴性患者的敏感性为18.4%(7/38)。 15例TB患者和14例其他皮肤病患者均无阳性。然而,在20个健康个体中只有1个是阳性,表明对培养滤液蛋白10(CFP-10)的抗体反应具有高度特异性(98.0%; 48/49)。从统计学上讲,发现基于CFP-10-的检测方法的性能可与抗酚糖脂I(PGL-1)抗体检测方法相媲美(P> 0.05)。因此,麻风分枝杆菌CFP-10潜在地是用于测量BI阳性麻风病患者的抗体应答的特异性抗原。 CFP-10是一种分泌性抗原,可作为宿主体内麻风分枝杆菌生存能力的标志物,因此其血清学潜力值得探索,可用于监测BI阳性麻风病患者(一种高度感染性形式)的反应)在化疗过程中。当比较细菌学和血清学结果时,82.1%的一致性表明,对麻风分枝杆菌CFP-10的血清阳性与细菌学标准非常吻合。因此,CFP-10似乎是将麻风病人分为BI阳性和BI阴性组的合适抗原。
  • 【亚氨基二琥珀酸差向异构酶的三维结构定义了MmgE / PrpD蛋白家族的折叠。】 复制标题 收藏 收藏
    DOI:10.1016/j.jmb.2006.07.051 复制DOI
    作者列表:Lohkamp B,Bäuerle B,Rieger PG,Schneider G
    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)差向异构酶催化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BY6对螯合剂亚氨基二琥珀酸酯的生物降解的一个步骤。该酶是MmgE / PrpD蛋白质家族的成员,MmgE / PrpD蛋白质家族是原核和真核来源的多种多样且鲜为特征的蛋白质。 IDS差向异构酶与已知三维结构的任何其他蛋白质均未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硒代蛋氨酸取代的酶通过多波长衍射确定为1.5 A的分辨率。在晶体中,酶形成同型二聚体,该亚基由两个结构域组成。较大的结构域,顺序不连续,包含残基Met1-Lys266和Leu400-Pro446,形成带有中心六螺旋束的新型全α螺旋折叠。第二个较小的域折叠成一个alpha beta域,该域在拓扑结构上与通过循环排列分支变异酶有关。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,其特征是带正电的表面,与带负电的底物(如亚氨基二琥珀酸酯)的结合相一致。对接实验提出了差向异构反应的两碱基机制。
  • 【与人神经胶质瘤有关的人核糖体蛋白L14.22的全长新基因。】 复制标题 收藏 收藏
    DOI: 复制DOI
    作者列表:Qi ZY,Hui GZ,Li Y,Zhou ZX,Gu SH,Xie Y
    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: 背景:本研究旨在通过cDNA微阵列技术获得与人神经胶质瘤相关的差异表达基因,并鉴定一个新的全长基因。
    方法:从人脑胶质瘤和正常脑组织中提取总RNA,并以mRNA为探针。杂交程序的结果用计算机系统扫描。随后通过Northern印迹,生物信息学方法和蛋白质表达来分析名为507E08视锥细胞的基因。
    结果:通过杂交和扫描4次,从人脑胶质瘤中获得了15个差异表达基因。 Northern印迹分析证实507E08克隆在人脑组织中低表达而在人脑胶质瘤组织中高表达。对BLASTn和BLASTx的分析表明,507E08克隆是一个新颖的全长基因,其编码蛋白质的203个氨基酸,被称为人核糖体蛋白质14.22基因。该核苷酸序列已提交给GenBank,登录号为AF329277。在大肠杆菌中表达后,蛋白质在SDS-PAGE凝胶上产生一条表观分子量为22 kDa的主带。
    结论:cDNA微阵列技术可成功用于鉴定差异表达的基因。人核糖体蛋白13.22的新的全长基因可能与人脑胶质瘤的发展有关。
  • 【苯巴比妥依赖和戒断大鼠脑中谷氨酸受体,c-fos mRNA表达和激活蛋白-1(AP-1)DNA结合活性的变化。】 复制标题 收藏 收藏
    DOI:10.1016/s0006-8993(97)00134-0 复制DOI
    作者列表:Tanaka S,Kiuchi Y,Numazawa S,Oguchi K,Yoshida T,Kuroiwa Y
    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)依赖和戒断大鼠大脑中谷氨酸受体的变化,立即早期基因的表达以及AP-1 DNA结合活性,以研究PB戒断综合征中谷氨酸受体活化的可能。通过喂食药物混合的食物5周来制备PB依赖的大鼠。放射自显影分析表明,结合物[3H()-5-甲基-10,11-二氢-5H-二苯并[a,d]环庚烯-5,10-亚胺e(MK-801)是一种N-甲基-拮抗剂D-天冬氨酸(NMDA)受体,在PB依赖和24小时戒断大鼠的大脑皮层中显着增加。然而,在海马中的[3H] MK-801结合以及在海马和大脑皮层中的[3H] 6-氰基-7-硝基喹喔啉-2,3-二酮(CNQX)和[3H]海藻酸结合基本上没有变化。组。 PB抽搐发作后,海马和大脑皮层中c-fos mRNA的表达增加,大脑皮层中c-jun mRNA的表达增加。通过施用MK-801抑制了c-fos和c-jun mRNA的诱导。此外,PB撤离可增强大脑中AP-1 DNA的结合活性。目前的发现表明,在PB戒断综合征的发展过程中,谷氨酸能神经传递的功能增强。

  • 【细胞外钙敏感受体的激活启动了朗格汉斯人胰岛的胰岛素分泌:蛋白激酶的参与。】 复制标题 收藏 收藏
    DOI:10.1677/joe.1.06891 复制DOI
    作者列表:Gray E,Muller D,Squires PE,Asare-Anane H,Huang GC,Amiel S,Persaud SJ,Jones PM
    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: :细胞外钙敏感受体(CaR)通常与全身性Ca(2)稳态相关,但该CaR在许多其他组织中也表达,包括Langerhans的胰岛。在本研究中,我们已经使用人类胰岛和胰岛素分泌细胞系(MIN6)来研究使用拟钙剂R-568(CaR激动剂在生理浓度的细胞外Ca(2)激活CaR的CaR激活)的作用。 。 CaR激活在葡萄糖的亚刺激浓度下启动了来自人胰岛和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。 CaR诱导的胰岛素分泌通过磷脂酶C或钙钙调蛋白依赖性激酶的抑制剂降低,但不通过蛋白激酶C抑制剂降低。 CaR激活还与p42 / 44丝裂原激活的蛋白激酶(MAPK)激活相关,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。
  • 【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。

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