• 【静止T细胞的有丝分裂刺激导致转录因子LSF迅速磷酸化,并增加了DNA结合活性。】 复制标题 收藏 收藏
    DOI:10.1101/gad.11.11.1435 复制DOI
    作者列表:Volker JL,Rameh LE,Zhu Q,DeCaprio J,Hansen U
    BACKGROUND & AIMS: The mammalian transcription factor LSF (CP2/LBP-1c) binds cellular promoters modulated by cell growth signals. We demonstrate here that LSF-DNA-binding activity is strikingly regulated by induction of cell growth in human peripheral T lymphocytes. Within 15 min of mitogenic stimulation of these cells, the level of LSF-DNA-binding activity increased by a factor of five. The level of LSF protein in the nucleus remained constant throughout this interval. However, a rapid decrease in the electrophoretic mobility of LSF, attributable to phosphorylation, correlated with the increase in DNA-binding activity. pp44 (ERK1) phosphorylated LSF in vitro on the same residue that was phosphorylated in vivo, specifically at amino acid position 291, as indicated by mutant analysis. As direct verification of the causal relationship between phosphorylation and DNA-binding activity, treatment in vitro of LSF with phosphatase both increased the electrophoretic mobility of the protein and decreased LSF-DNA-binding activity. This modulation of LSF-DNA-binding activity as T cells progress from a resting to a replicating state reveals that LSF activity is regulated during cell growth and suggests that LSF regulates growth-responsive promoters.

    背景与目标: 哺乳动物转录因子LSF(CP2 / LBP-1c)结合受细胞生长信号调节的细胞启动子。我们在这里证明,LSF-DNA结合活性是由人类外周血T淋巴细胞的细胞生长诱导来显着调节的。在这些细胞的促有丝分裂刺激的15分钟内,LSF-DNA结合活性的水平提高了五倍。在整个间隔期间,核中LSF蛋白的水平保持恒定。但是,可归因于磷酸化的LSF电泳迁移率的快速下降与DNA结合活性的增加有关。突变分析表明,pp44(ERK1)在体外磷酸化的同一残基上,特别是在氨基酸位置291处磷酸化LSF。作为磷酸化与DNA结合活性之间因果关系的直接验证,体外用磷酸酶处理LSF既增加了蛋白质的电泳迁移率,又降低了LSF-DNA结合活性。当T细胞从静止状态发展到复制状态时,对LSF-DNA结合活性的这种调节揭示了LSF活性在细胞生长过程中受到调节,并暗示LSF调节生长响应性启动子。

  • 【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)。本研究的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,在21名患者中有11名患者(52%)对依那西普治疗有反应,包括6名患者(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,尤其是在胃肠道受累的情况下。
  • 【在第3天或第5天胚胎移植后测量血清雌二醇和β-HCG,以解释妊娠结局。】 复制标题 收藏 收藏
    DOI:10.1016/s1472-6483(10)60631-1 复制DOI
    作者列表:Kumbak B,Oral E,Karlikaya G,Lacin S,Kahraman S
    BACKGROUND & AIMS: :The aim of this study was to assess the clinical value of serum oestradiol concentration 8 days after embryo transfer (D8E2) and beta-human chorionic gonadotrophin (HCG-beta) concentration 12 days after embryo transfer (D12HCG-beta) in the prediction of pregnancy and the outcome of pregnancy following assisted reproduction, taking into account the day of transfer, which was either day 3 (D3) or day 5 (D5). The objective was to improve patient counselling by giving quantitative and reliable predictive information instead of non-specific uncertainties. A total of 2035 embryo transfer cycles performed between January 2003 and June 2005 were analysed retrospectively. Biochemical pregnancy, ectopic pregnancy and first-trimester abortions were classified as non-viable pregnancies; pregnancies beyond 12 weeks gestation were classified as ongoing pregnancies (OP). Significantly higher D8E2 and D12HCG-beta were obtained in D5 transfers compared with D3 transfers with regard to pregnancy and OP (P
    背景与目标: :这项研究的目的是评估胚胎移植后8天(D8E2)和β-人绒毛膜促性腺激素(HCG-beta)浓度在胚胎移植12天后(D12HCG-beta)的临床价值,以预测糖尿病的发生。怀孕和辅助生殖后的怀孕结局,要考虑到转移的那天,即第3天(D3)或第5天(D5)。目的是通过提供定量和可靠的预测信息而不是非特定的不确定性来改善患者咨询。回顾性分析了2003年1月至2005年6月进行的2035个胚胎移植周期。生化妊娠,异位妊娠和早孕流产被归类为不可行的妊娠。妊娠超过12周的孕妇被分类为进行中的孕妇(OP)。与妊娠和OP的D3转移相比,D5转移获得的D8E2和D12HCG-beta明显更高(P <或= 0.001)。对于D3胚胎移植,D8E2预测OP的临界值为130 pg / ml(敏感性80%,特异性72%),而D12HCG-beta为98 mIU / ml(敏感性89%,特异性69%)。对于D5胚胎移植,该值分别为179 pg / ml(敏感性为79%,特异性为84%)和257 mIU / ml(敏感性为78%,特异性为81%)。看来,胚胎移植后血清D8E2和D12HCG-β的浓度提供了有关妊娠以及IVF胚胎移植后妊娠结局的明确信息。
  • 【角质形成细胞迁移和肽生长因子:PDGF,bFGF,EGF,IGF-I,aFGF和TGF-β对胶原凝胶中人角膜细胞迁移的影响。】 复制标题 收藏 收藏
    DOI:10.1076/ceyr.16.6.605.5081 复制DOI
    作者列表:Andresen JL,Ledet T,Ehlers N
    BACKGROUND & AIMS: PURPOSE:Peptide growth factors are known accelerators of corneal wound healing, probably mediated through increased proliferation of the cells; however, information about their effect on keratocyte motility is lacking. The influence of peptide growth factors on keratocyte migratory activity was investigated, using the following growth factors: platelet derived growth factor (PDGF-BB), epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and transforming growth factor-beta-1 (TGF-beta 1).

    METHODS:Keratocytes were seeded on gels of type 1 collagen, growth factor added, and the cells left to migrate for 72 hours. Subsequently, the number of keratocytes at the different levels in the collagen gel was evaluated by optically sectioning the gel at 20 microns, intervals, with an inverted phase contrast microscope.

    RESULTS:PDGF, EGF and bFGF at 10 ng/ml, all increased the number of keratocytes at the different levels of the gel as compared to a non-stimulated control (p < 0.05 or p < 0.01, students t-test). TGF-beta proved to be a strong inhibitor of keratocyte migration, decreasing the number of keratocytes observed at every level in the gel (p < 0.05 and p < 0.01, students t-test), whereas no effect of IGF-I and aFGF was found. During the 72 hours of migration, no contraction of the collagen gels was observed. Autoradiography of histological sections of the gels showed that during the 72-hour period only TGF-beta and 10% fetal bovine serum induced an increase in keratocyte proliferation.

    CONCLUSION:PDGF, EGF and bFGF increase keratocyte migration, independent of proliferation in a collagen gel invasion assay and might promote corneal wound healing, not only by increasing cell proliferation, but also through increased motility.

    背景与目标: 目的:肽生长因子是角膜伤口愈合的已知促进剂,可能是通过细胞增殖的增加来介导的。然而,缺乏有关它们对角膜细胞运动性影响的信息。使用以下生长因子研究了肽生长因子对角膜细胞迁移活性的影响:血小板衍生生长因子(PDGF-BB),表皮生长因子(EGF),酸性成纤维细胞生长因子(aFGF),碱性成纤维细胞生长因子(bFGF) ),胰岛素样生长因子I(IGF-I)和转化生长因子β-1(TGF-beta 1)。

    方法:将角质形成细胞接种在1型胶原蛋白,添加了生长因子,细胞迁移了72小时。随后,通过倒置相差显微镜以20微米的间隔对凝胶进行光学切片,评估胶原蛋白凝胶中不同水平的角质形成细胞的数量。

    结果:PDGF与未刺激的对照组相比,10 ng / ml的EGF和bFGF均增加了凝胶水平不同时的角膜细胞数量(p <0.05或p <0.01,学生t检验)。 TGF-β被证明是一种强烈的角膜细胞迁移抑制剂,可减少凝胶中各个水平上观察到的角膜细胞数量(p <0.05和p <0.01,学生t检验),而IGF-I和aFGF则没有作用成立。在迁移的72小时内,未观察到胶原凝胶的收缩。凝胶组织学切片的放射自显影显示,在72小时内,只有TGF-β和10%的胎牛血清诱导了角膜细胞增殖的增加。

    结论:PDGF,EGF bFGF和bFGF可增加角膜细胞迁移,而不受胶原凝胶入侵试验中的增殖的影响,并且不仅可以通过增加细胞增殖,而且可以通过增加运动性来促进角膜伤口愈合。

  • 【Xanthomonas campestris pv的特异结合。 vesicatoria AraC型转录激活因子HrpX到植物诱导型启动子盒。】 复制标题 收藏 收藏
    DOI:10.1128/JB.00795-06 复制DOI
    作者列表:Koebnik R,Krüger A,Thieme F,Urban A,Bonas U
    BACKGROUND & AIMS: :The pathogenicity of the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion system which is encoded by the 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Expression of the hrp operons is strongly induced in planta and in a special minimal medium and depends on two regulatory proteins, HrpG and HrpX. In this study, DNA affinity enrichment was used to demonstrate that the AraC-type transcriptional activator HrpX binds to a conserved cis-regulatory element, the plant-inducible promoter (PIP) box (TTCGC-N(15)-TTCGC), present in the promoter regions of four hrp operons. No binding of HrpX was observed when DNA fragments lacking a PIP box were used. HrpX also bound to a DNA fragment containing an imperfect PIP box (TTCGC-N(8)-TTCGT). Dinucleotide replacements in each half-site of the PIP box strongly decreased binding of HrpX, while simultaneous dinucleotide replacements in both half-sites completely abolished binding. Based on the complete genome sequence of Xanthomonas campestris pv. vesicatoria, putative plant-inducible promoters consisting of a PIP box and a -10 promoter motif were identified in the promoter regions of almost all HrpX-activated genes. Bioinformatic analyses and reverse transcription-PCR experiments revealed novel HrpX-dependent genes, among them a NUDIX hydrolase gene and several genes with a predicted role in the degradation of the plant cell wall. We conclude that HrpX is the most downstream component of the hrp regulatory cascade, which is proposed to directly activate most genes of the hrpX regulon via binding to corresponding PIP boxes.
    背景与目标: :植物致病性细菌Xanthomonas campestris pv的致病性。 vesicatoria依赖于由23-kb hrp(过敏反应和致病性)基因簇编码的III型分泌系统。 hrp操纵子的表达在植物和特殊的基本培养基中强烈诱导,并依赖于两种调节蛋白HrpG和HrpX。在这项研究中,DNA亲和力富集用于证明AraC型转录激活因子HrpX与保守的顺式调控元件,即植物诱导型启动子(PIP)框(TTCGC-N(15)-TTCGC)结合,四个hrp操纵子的启动子区域。当使用缺少PIP盒的DNA片段时,未观察到HrpX的结合。 HrpX还绑定到包含不完整的PIP盒(TTCGC-N(8)-TTCGT)的DNA片段。 PIP盒每个半位中的二核苷酸替换强烈降低了HrpX的结合,而两个半位中同时进行的二核苷酸替换则完全消除了结合。基于Xanthomonas campestris pv的完整基因组序列。在几乎所有的HrpX激活基因的启动子区域中,鉴定出了由vesptoria组成的假定的植物诱导型启动子,该启动子由PIP框和-10个启动子组成。生物信息学分析和逆转录-PCR实验揭示了新的HrpX依赖性基因,其中包括NUDIX水解酶基因和几个在植物细胞壁降解中具有预测作用的基因。我们得出的结论是,HrpX是hrp调节级联的最下游组件,建议通过与相应的PIP盒结合直接激活hrpX regulon的大多数基因。
  • 【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用于监测BI阳性麻风病患者(高度感染性)的反应中。 )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人知的蛋白质。 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,以研究谷氨酸受体激活在PB戒断综合征中的可能。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,[3H()-5-甲基-10,11-二氢-5H-二苯并[a,d]环庚烯-5,10-亚胺e(MK-801)与N-甲基- 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之前和期间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。
  • 【烟碱乙酰胆碱受体的酪氨酸磷酸化介导了Grb2结合。】 复制标题 收藏 收藏
    DOI: 复制DOI
    作者列表:Colledge M,Froehner SC
    BACKGROUND & AIMS: Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) is associated with an altered rate of receptor desensitization and also may play a role in agrin-induced receptor clustering. We have demonstrated a previously unsuspected interaction between Torpedo AChR and the adaptor protein Grb2. The binding is mediated by the Src homology 2 (SH2) domain of Grb2 and the tyrosine-phosphorylated delta subunit of the AChR. Dephosphorylation of the delta subunit abolishes Grb2 binding. A cytoplasmic domain of the delta subunit contains a binding motif (pYXNX) for the SH2 domain of Grb2. Indeed, a phosphopeptide corresponding to this region of the delta subunit binds to Grb2 SH2 fusion proteins with relatively high affinity, whereas a peptide lacking phosphorylation on tyrosine exhibits no binding. Grb2 is colocalized with the AChR on the innervated face of Torpedo electrocytes. Furthermore, Grb2 specifically copurifies with AChR solubilized from postsynaptic membranes. These data suggest a novel role for tyrosine phosphorylation of the AChR in the initiation of a Grb2-mediated signaling cascade at the postsynaptic membrane.

    背景与目标: 烟碱样乙酰胆碱受体(AChR)的酪氨酸磷酸化与受体脱敏率的改变有关,也可能在凝集素诱导的受体簇中起作用。我们已经证明了鱼雷AChR和衔接蛋白Grb2之间以前没有想到的相互作用。结合是由Grb2的Src同源2(SH2)域和AChR的酪氨酸磷酸化的δ亚基介导的。 δ亚基的去磷酸化消除了Grb2结合。 δ亚基的胞质结构域包含Grb2的SH2结构域的结合基序(pYXNX)。实际上,对应于δ亚基的该区域的磷酸肽以相对高的亲和力与Grb2 SH2融合蛋白结合,而在酪氨酸上缺乏磷酸化的肽没有结合。 Grb2与AChR在鱼雷电池的神经支配面上共定位。此外,Grb2与从突触后膜溶解的AChR特异性共纯化。这些数据表明,AChR的酪氨酸磷酸化在突触后膜的Grb2介导的信号级联反应的起始中起着新的作用。

  • 【鸡GATA-2和GATA-3的N末端指是独立的序列特异性DNA结合结构域。】 复制标题 收藏 收藏
    DOI:10.1093/emboj/16.10.2874 复制DOI
    作者列表:Pedone PV,Omichinski JG,Nony P,Trainor C,Gronenborn AM,Clore GM,Felsenfeld G
    BACKGROUND & AIMS: The GATA family of vertebrate DNA binding regulatory proteins are expressed in diverse tissues and at different times of development. However, the DNA binding regions of these proteins possess considerable homology and recognize a rather similar range of DNA sequence motifs. DNA binding is mediated through two domains, each containing a zinc finger. Previous results have led to the conclusion that although in some cases the N-terminal finger can contribute to specificity and strength of binding, it does not bind independently, whereas the C-terminal finger is both necessary and sufficient for binding. Here we show that although this is true for the N-terminal finger of GATA-1, those of GATA-2 and GATA-3 are capable of strong independent binding with a preference for the motif GATC. Binding requires the presence of two basic regions located on either side of the N-terminal finger. The absence of one of these near the GATA-1 N-terminal finger probably accounts for its inability to bind. The combination of a single finger and two basic regions is a new variant of a motif that has been previously found in the binding domains of other finger proteins. Our results suggest that the DNA binding properties of the N-terminal finger may help distinguish GATA-2 and GATA-3 from GATA-1 and the other GATA family members in their selective regulatory roles in vivo.

    背景与目标: GATA家族的脊椎动物DNA结合调节蛋白在不同的组织中以及在不同的发育时期表达。但是,这些蛋白质的DNA结合区具有相当的同源性,可以识别相当相似范围的DNA序列基序。 DNA结合是通过两个结构域介导的,每个结构域都包含一个锌指。先前的结果得出的结论是,尽管在某些情况下N末端指可以有助于结合的特异性和强度,但它不能独立地结合,而C末端指对于结合既是必需的又是足够的。在这里,我们表明,尽管对于GATA-1的N端手指来说确实如此,但GATA-2和GATA-3的手指却能够强烈独立地结合,并优先选择基序GATC。绑定需要在N末端指状体的两侧都存在两个基本区域。在GATA-1 N端手指附近缺少这些手指之一可能是其无法结合的原因。单个手指和两个基本区域的组合是一个基序的新变体,该变体先前已在其他手指蛋白的结合域中发现。我们的结果表明,N末端手指的DNA结合特性可能有助于区分GATA-2和GATA-3与GATA-1和其他GATA家族成员在体内的选择性调节作用。

  • 【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)被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)活化和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)迅速而惊人的激活,这是通过体外激酶测定和激活环(Ser706 / 710)和自磷酸化位点(Ser876)的磷酸化测量的。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。

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