• 【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。
  • 【缺氧条件下的肿瘤基质细胞相互作用通过肝细胞生长因子/ c-Met途径增加了胰腺癌细胞的侵袭性。】 复制标题 收藏 收藏
    DOI:10.1002/ijc.22178 复制DOI
    作者列表:Ide T,Kitajima Y,Miyoshi A,Ohtsuka T,Mitsuno M,Ohtaka K,Koga Y,Miyazaki K
    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。基质细胞与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,我们调查了低氧刺激是否影响基质和胰腺癌细胞。我们的研究结果表明,缺氧显着提高了胰腺癌(PK8)和成纤维细胞(MRC5)中HIF-1alpha的表达。低氧刺激加速了PK8细胞的侵袭活性,因此,当用由低氧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,侵袭性进一步加快。在缺氧条件下,PK8细胞中的MMP-2,MMP-7,MT1-MMP和c-Met表达增加。缺氧刺激还增加了MRC5细胞的肝细胞生长因子(HGF)分泌,这导致PK8细胞中c-Met磷酸化的升高。相反,通过从低氧条件培养基中除去HGF,可以降低PK8细胞的癌浸润,MMP活性和c-Met磷酸化升高。在免疫组织化学研究中,在周围的基质细胞和胰腺癌细胞中均观察到了HIF-1alpha的表达,因此表明在癌细胞和基质细胞中均存在缺氧。此外,发现基质HGF表达不仅与癌细胞中的基质HIF-1α表达而且与c-Met表达显着相关。这些结果表明基质以及癌细胞内的低氧环境激活了HGF / c-Met系统,从而促进了胰腺癌的侵袭性侵袭性特征。
  • 【促胰液素PulD的C末端结构域包含其同源伴侣PulS的结合位点,并赋予PulS对pIVf1功能的依赖性。】 复制标题 收藏 收藏
    DOI:10.1046/j.1365-2958.1997.3531727.x 复制DOI
    作者列表:Daefler S,Guilvout I,Hardie KR,Pugsley AP,Russel M
    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,称为促胰液素,参与许多革兰氏阴性细菌的外膜大分子的分泌。在支链淀粉酶分泌系统中,PulS是一种与外膜相关的脂蛋白,是PulD促胰液素的完整性和正确的外膜定位所必需的。在这里,我们显示PulS结合位点位于PulD的C末端65个残基内。此结构域添加到丝状噬菌体分泌蛋白,pIV或无关的麦芽糖结合蛋白上,使得这两种蛋白都依赖于PulS来保持稳定性。由噬菌体f1 pIV和PuID的C末端结构域组成的嵌合蛋白需要正确定位PulS以支持噬菌体装配。通过共免疫沉淀法和亲和色谱法检测到了pIV-PulD65嵌合体和PulS之间形成的体内复合物。
  • 【白色念珠菌myristoylCoA的选择性肽和拟肽抑制剂:蛋白N-肉豆蔻酰基转移酶:一种抗真菌治疗的新方法。】 复制标题 收藏 收藏
    DOI:10.1002/(SICI)1097-0282(1997)43:1<43::AID-BIP5>3.0 复制DOI
    作者列表:Sikorski JA,Devadas B,Zupec ME,Freeman SK,Brown DL,Lu HF,Nagarajan S,Mehta PP,Wade AC,Kishore NS,Bryant ML,Getman DP,McWherter CA,Gordon JI
    BACKGROUND & AIMS: MyristoylCoA:protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachment of a rare cellular fatty acid, myristate, to the N-terminal Gly residue of a variety of eukaryotic proteins. The myristoyl moiety is often essential for expression of the biological functions for these proteins.

    Attachment of C14:0 alone provides barely enough hydrophobicity to allow stable association with membranes. The partitioning of N-myrisotylproteins is therefore often modulated by "switches" that function through additional covalent or noncovalent modifications. Candida albicans, the principal cause of systemic fungal infection in immunocompromised humans, contains a single NMT gene that is essential for its viability. The functional properties of the acylCoA binding site of human and C. albicans NMT are very similar. However, there are distinct differences in their peptide binding sites. An ADP ribosylation factor (Arf) is included among the few cellular protein substrates of the fungal enzyme. Alanine scanning mutagenesis of an octapeptide derived from an N-terminal Arf sequence (GLYASKLS-NH2) disclosed that Gly1, Ser5, and Lys6 play predominant roles in binding. ALYASKLS-NH2 is an inhibitor competitive for peptide [Ki(app) = 15.3 +/- 6.4 microM] and noncompetitive for myristoylCoA. Remarkably, replacement of the N-terminal tetrapeptide with an 11-aminoundecanoyl group results in a competitive inhibitor (11-aminoundecanoyl-SKLS-NH2) that is approximately 40-fold more potent [Ki(app) = 0.40 +/- 0.03 microM] than the starting octapeptide. Removal of Leu-Ser from the C-terminus generates a competitive dipeptide inhibitor (11-aminoundecanoyl-SK-NH2) with a Ki(app) of 11.7 +/- 0.4 microM, equivalent to that of the starting octapeptide. A derivative dipeptide inhibitor containing a C-terminal N-cyclohexylethyl lysinamide moiety has the advantage of being more potent (IC50 = 0.11 +/- 0.03 microM) and resistant to digestion by cellular carboxypeptidases. Rigidifying the flexible aminoundecanoyl chain results in very potent general NMT inhibitors (IC50 = 40-50 nM). Substituting a 2-methylimidazole for the N-terminal amine and adding a benzylic alpha-methyl group with R stereochemistry to the rigidifying element produces even more potent inhibitors (IC50 = 20-50 nM) that are up to 500-fold selective for the fungal compared to human enzyme. A related less potent member of this series of compounds is fungistatic. Its growth inhibitory effects are associated with a reduction in cellular protein N-myristoylation, judged using cellular Arf as a reporter. These studies establish that NMT is a new antifungal target.

    背景与目标: MyristoylCoA :蛋白N-肉豆蔻酰基转移酶(NMT)催化稀有细胞脂肪酸肉豆蔻酸酯与多种真核蛋白N-末端Gly残基的共翻译共价连接。肉豆蔻酰基部分通常对于表达这些蛋白质的生物学功能是必不可少的。

    仅C14 :0的附件仅提供了不足以使与膜稳定结合的疏水性。因此,N-肉豆蔻酰蛋白的分区通常由通过附加的共价或非共价修饰起作用的“开关”调节。白色念珠菌是免疫力低下的人体内全身真菌感染的主要原因,它包含一个对其生存能力至关重要的单个NMT基因。人和白色念珠菌NMT的酰基辅酶A结合位点的功能特性非常相似。但是,它们的肽结合位点存在明显差异。 ADP核糖基化因子(Arf)包括在真菌酶的几种细胞蛋白底物中。来自N末端Arf序列(GLYASKLS-NH2)的八肽的丙氨酸扫描诱变显示,Gly1,Ser5和Lys6在结合中起主要作用。 ALYASKLS-NH2是一种对肽[Ki(app)= 15.3 /-6.4 microM]具有竞争性的抑制剂,对肉豆蔻酰辅酶A不具有竞争性。值得注意的是,用11-氨基十一烷酰基取代N末端四肽会产生竞争性抑制剂(11-氨基十一烷酰基-SKLS-NH2),其效力比[Ki(app)= 0.40 /-0.03 microM]高40倍左右。起始八肽。从C末端去除Leu-Ser会产生竞争性的二肽抑制剂(11-氨基十一烷酰基-SK-NH2),Ki(app)为11.7 /-0.4 microM,与起始八肽相当。含有C-末端N-环己基乙基赖氨酰胺部分的衍生物二肽抑制剂具有更有效的优势(IC 50 = 0.11 /0.03μM),并且对细胞羧肽酶的消化具有抵抗力。刚性化柔性氨基十一烷酰基链会产生非常有效的常规NMT抑制剂(IC50 = 40-50 nM)。用2-甲基咪唑代替N端胺,并在刚性元素中添加具有R立体化学的苄基α-甲基,可产生更强效的抑制剂(IC50 = 20-50 nM),对真菌的选择性高达500倍与人类酶相比。该系列化合物的一个相关的效力较弱的成员是抑真菌的。使用细胞Arf作为报告基因判断,其生长抑制作用与细胞蛋白N-肉豆蔻酰化的减少有关。这些研究确定NMT是一种新的抗真菌靶标。

  • 【纤连蛋白与成纤维细胞和纤连蛋白的III1组件结合所需的N端I型模块。】 复制标题 收藏 收藏
    DOI:10.1042/bj3230051 复制DOI
    作者列表:Sottile J,Mosher DF
    BACKGROUND & AIMS: Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conformation-dependent binding site for the 70 kDa N-terminal region of fibronectin, suggesting that the III1 module on cell-surface fibronectin may serve as a binding site for fibronectin's N-terminus on substrate-attached cells. To explore this possiblility, we compared the ability of mutant recombinant 70 kDa proteins containing deletions of one or several of the first five type I modules to bind to fibroblasts and to III1. Proteins containing the fourth and fiftBiomolecular Chemistry and Medicine, University of Wisconsin, Madison, WI 53706U.S.A. Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conh as 70 kDa deletion mutants lacking I4 and I5 also bound to the cell surface, and deletion mutants lacking I1-3 and I4-5 both competed only partially for binding of 125I-labelled fibronectin or 70 kDa protein. These data indicate that the N-terminal part of fibronectin binds to III1 via I4 and I5 and that interactions in addition to that of I4 and I5 with III1 are important for cell-surface-mediated fibronectin polymerization.

    背景与目标: 纤连蛋白原纤维的组装发生在附着有底物的细胞表面,并由分子的N端70 kDa部分中的第一至第五类I模块介导。纤连蛋白的第一个III型模块(III1)(不存在于70 kDa的部分中)包含纤连蛋白70 kDa N端区域的构象依赖性结合位点,表明细胞表面纤连蛋白上的III1模块可以作为底物附着细胞上纤连蛋白N末端的结合位点。为了探讨这种可能性,我们比较了包含前五个I型模块中一个或多个缺失的突变重组70 kDa蛋白与成纤维细胞和III1结合的能力。含有第四和第五种蛋白质的蛋白质生物分子化学和医学,威斯康星大学麦迪逊分校,威斯康星州53706纤连蛋白原纤维的组装发生在附着有底物的细胞表面,并由分子的N端70 kDa部分中的第一至第五类I模块介导。纤连蛋白的第一个III型模块(III1)(不存在于70 kDa的部分)包含一个conh,因为缺少I4和I5的70 kDa缺失突变体也与细胞表面结合,而缺少I1-3和I4-5的缺失突变体都存在仅部分竞争结合125 I标记的纤连蛋白或70 kDa蛋白。这些数据表明纤连蛋白的N末端部分通过I4和I5与III1结合,并且除了I4和I5之外,与III1的相互作用对于细胞表面介导的纤连蛋白聚合也很重要。
  • 【N末端甘氨酸对流感血凝素融合肽与脂质双层的二级结构,方向和相互作用的影响。】 复制标题 收藏 收藏
    DOI:10.1016/S0006-3495(96)79793-X 复制DOI
    作者列表:Gray C,Tatulian SA,Wharton SA,Tamm LK
    BACKGROUND & AIMS: The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.

    背景与目标: 流感血凝素(HA)的膜锚定亚基的氨基末端片段在膜融合中起着至关重要的作用,因此被称为融合肽。我们已经通过荧光,圆二色性和傅里叶变换研究了对应于野生型和几个融合和非融合突变体的流感病毒HA融合肽的N末端改变的二级肽的二级结构,方向及其对双层肽结构的影响。红外光谱。所有肽都包含α-螺旋和β-链构象的区段。在野生型融合肽中,所有残基的40%在α-二级结构中,而30%在β-二级结构中。相比之下,非融合肽表现出较大的β/α二级结构比。基于各个吸收带的红外二色性,分别测量野生型融合肽的β链的螺旋和酰胺羰基的有序参数。对于野生型肽的两个片段,发现有序参数在0.1-0.7范围内,这表明它们最有可能以与膜法线倾斜的角度排列。非融合肽而非融合肽诱导了1735 cm(-1)处的红外吸收带的分裂,这被分配给了脂质酯羰基键的拉伸振动。该分裂报告了脂酯羰基与融合肽的水和/或供氢基团之间形成的氢键的改变,与肽的β/α比率相关,表明未配对的β链可能取代水分子,并与脂质酯的羰基氢键合。融合肽极端N端由单个氨基酸置换引起的深刻结构变化进一步表明,当融合肽结合脂质双层时,三级或四级结构相互作用可能很重要。

  • 【淋巴系统及其特异性生长因子,血管内皮生长因子C在前列腺癌的淋巴转移中的作用。】 复制标题 收藏 收藏
    DOI:10.1111/j.1464-410X.2006.06403.x 复制DOI
    作者列表:Trojan L,Rensch F,Voss M,Grobholz R,Weiss C,Jackson DG,Alken P,Michel MS
    BACKGROUND & AIMS: OBJECTIVE:To compare prostate carcinoma, with and with no lymph node metastasis, to benign prostatic hyperplasia (BPH) tissue for lymphatic vessel density (LVD) and the expression of the lymph-endothelial specific growth factor, vascular endothelial growth factor C (VEGF-C), to determine their role in lymphogenic metastasis. PATIENTS, MATERIALS AND METHODS:Lymphatic vessels were stained using lymphatic vessel endothelial hyaluronan receptor 1 and assessed in standard areas. The expression of VEGF-C was assessed by the number of positive epithelial cells. The data were compared with the clinical staging. RESULTS:The lowest LVD was found in tumorous areas as opposed to periphery and nontumorous tissue (P = 0.007; P < 0.001). The highest LVD was in BPH tissue (P < 0.001). There was no correlation with clinical staging. There was more VEGF-C staining in pN1 than in pN0 and in BPH specimens (P = 0.002). CONCLUSION:LVD is not a prognostic variable for the process of lymphogenic metastasis in prostate cancer. VEGF-C is up-regulated in prostate cancer and its correlation with lymph node status suggests a role for the development of lymph node metastasis, e.g. via an increased permeability of lymphatic vessels.
    背景与目标: 目的:比较有无淋巴结转移的前列腺癌与良性前列腺增生(BPH)组织的淋巴管密度(LVD)以及淋巴内皮特异性生长因子,血管内皮生长因子C(VEGF- C),确定其在淋巴转移中的作用。
    患者,材料和方法:使用淋巴管内皮透明质酸受体1对淋巴管进行染色,并在标准区域进行评估。通过阳性上皮细胞的数量评估VEGF-C的表达。将数据与临床分期进行比较。
    结果:与周围和非肿瘤组织相比,在肿瘤区域发现的LVD最低(P = 0.007; P <0.001)。 LVD最高的是BPH组织(P <0.001)。与临床分期无关。 pN1和BPH标本中的VEGF-C染色要多于pN0和PPH(P = 0.002)。
    结论:LVD不是前列腺癌淋巴转移的预后变量。 VEGF-C在前列腺癌中被上调,并且其与淋巴结状态的相关性暗示了淋巴结转移的发展,例如肝癌的发生。通过增加淋巴管的通透性
  • 【用于屈光手术时丝裂霉素C的全身吸收。】 复制标题 收藏 收藏
    DOI:10.1016/j.jcrs.2012.08.062 复制DOI
    作者列表:Crawford C,Ainbinder DJ,Davis R,George RK,Rivers B,Wingerd MA,Torres M,Dent A
    BACKGROUND & AIMS: PURPOSE:To determine whether corneal topical application of mitomycin-C (MMC) results in measurable plasma levels of systemic absorption. SETTING:Madigan Army Medical Center, Refractive Surgery Center, Fort Lewis, Washington, and Micro-Constants Laboratory, San Diego, California, USA. DESIGN:Case-control study. METHODS:The study comprised male and female active-duty soldiers having excimer laser photorefractive keratectomy with MMC. Patients who met inclusion criteria were asked to provide a blood sample immediately after being treated with MMC 0.2 mg/mL (0.02%) for 30 seconds. Human plasma samples were evaluated by liquid chromatography mass spectrometry to determine whether MMC was present. RESULTS:Thirty samples were submitted for evaluation. There was zero detection of MMC in the submitted samples. The quantifiable limit was greater than 10.0 ng/mL. All samples were below this. CONCLUSIONS:In this study of 30 patients with topical application of MMC for refractive surgery, there was no measurable evidence of systemic absorption. Although systemic absorption has been found with use in larger quantities, it was not known whether MMC toxicity concerns could be extrapolated to the refractive surgery population. This information allows counseling of patients on the extremely low likelihood of systemic absorption or toxicity following current techniques for refractive surgery. FINANCIAL DISCLOSURE:No author has a financial or proprietary interest in any material or method mentioned.
    背景与目标: 目的:确定局部应用丝裂霉素-C(MMC)的角膜是否可测量血浆中的全身吸收水平。
    地点:华盛顿州刘易斯堡的马迪根军医中心,屈光手术中心和美国加利福尼亚圣地亚哥的微常数实验室。
    设计:病例对照研究。
    方法:该研究包括接受MMC准分子激光屈光性角膜切除术的现役士兵。接受入选标准的患者接受MMC 0.2 mg / mL(0.02%)治疗30秒后立即提供血样。通过液相色谱质谱法评估人血浆样品,以确定是否存在MMC。
    结果:提交了30个样品进行评估。在提交的样本中,MMC的检测为零。定量限大于10.0 ng / mL。所有样品均低于此值。
    结论:在这项对30例MMC局部应用屈光手术的患者的研究中,没有可测量的全身吸收证据。尽管已发现全身吸收的使用量更大,但尚不清楚是否可以将MMC毒性问题推断到屈光手术人群中。该信息使患者可以根据当前屈光手术技术对全身吸收或毒性的可能性极低进行咨询。
    财务披露:任何作者都不会对所提及的任何材料或方法具有财务或专有利益。
  • 【通过核酸碱基的四分之一化与芳香族氨基酸的突出堆积相互作用:X射线晶体学特征和生物学意义。】 复制标题 收藏 收藏
    DOI:10.1016/0003-9861(90)90251-s 复制DOI
    作者列表:Ishida T,Ueda H,Segawa K,Doi M,Inoue M
    BACKGROUND & AIMS: :In order to investigate the mode of interaction between the N-quarternized cytosine base and the aromatic amino acid, the crystal structure of the 3-methyl-cytidine-5'-monophosphate:tryptamine complex was analyzed by X-ray diffraction. The complex crystals were stabilized by extensive hydrogen bond formations in which eight independent water molecules per complex pair participated. A prominent stacking interaction, characterized by a parallel alignment of both rings with a separation distance of ca. 3.4 A, was observed between the cytosine base and the indole ring. Combining the present results with X-ray crystallographic data on the adenine--and guanine--aromatic amino acid interactions, we summarize the structural characteristics observed in the stacking interaction of the N-quarternized nucleic acid base with the aromatic amino acid and discuss their biological implications, especially in connection with the significance of N-protonation of nucleic acid base for selective recognition by protein.
    背景与目标: :为了研究N-季铵化的胞嘧啶碱基与芳族氨基酸之间的相互作用方式,通过X射线衍射分析了3-甲基胞苷-5'-单磷酸酯:色胺的配合物的晶体结构。复杂的晶体通过广泛的氢键形成而稳定,其中每个复杂对参与八个独立的水分子。突出的堆叠相互作用,其特征在于两个环的平行排列的间隔距离为ca。在胞嘧啶碱基和吲哚环之间观察到3.4A。将当前结果与腺嘌呤-鸟嘌呤-芳族氨基酸相互作用的X射线晶体学数据相结合,我们总结了在N-季铵化核酸碱基与芳族氨基酸的堆叠相互作用中观察到的结构特征,并讨论了它们的结构特征。生物学意义,特别是与核酸碱基的N质子化对于蛋白质选择性识别的意义有关。
  • 【遗传,结构和化学方面的见解,揭示了精子发生过程中GRASP55在生殖细胞高尔基体重塑和JAM-C极化定位中的双重功能。】 复制标题 收藏 收藏
    DOI:10.1371/journal.pgen.1006803 复制DOI
    作者列表:Cartier-Michaud A,Bailly AL,Betzi S,Shi X,Lissitzky JC,Zarubica A,Sergé A,Roche P,Lugari A,Hamon V,Bardin F,Derviaux C,Lembo F,Audebert S,Marchetto S,Durand B,Borg JP,Shi N,Morelli X,Aurrand-Lions M
    BACKGROUND & AIMS: :Spermatogenesis is a dynamic process that is regulated by adhesive interactions between germ and Sertoli cells. Germ cells express the Junctional Adhesion Molecule-C (JAM-C, encoded by Jam3), which localizes to germ/Sertoli cell contacts. JAM-C is involved in germ cell polarity and acrosome formation. Using a proteomic approach, we demonstrated that JAM-C interacted with the Golgi reassembly stacking protein of 55 kDa (GRASP55, encoded by Gorasp2) in developing germ cells. Generation and study of Gorasp2-/- mice revealed that knock-out mice suffered from spermatogenesis defects. Acrosome formation and polarized localization of JAM-C in spermatids were altered in Gorasp2-/- mice. In addition, Golgi morphology of spermatocytes was disturbed in Gorasp2-/- mice. Crystal structures of GRASP55 in complex with JAM-C or JAM-B revealed that GRASP55 interacted via PDZ-mediated interactions with JAMs and induced a conformational change in GRASP55 with respect of its free conformation. An in silico pharmacophore approach identified a chemical compound called Graspin that inhibited PDZ-mediated interactions of GRASP55 with JAMs. Treatment of mice with Graspin hampered the polarized localization of JAM-C in spermatids, induced the premature release of spermatids and affected the Golgi morphology of meiotic spermatocytes.
    背景与目标: :生精是一个动态过程,受生殖细胞和支持细胞之间的粘附相互作用调节。生殖细胞表达连接黏附分子-C(JAM-C,由Jam3编码),其位于细菌/ Sertoli细胞接触处。 JAM-C与生殖细胞极性和顶体形成有关。使用蛋白质组学方法,我们证明了JAM-C与发育中的生殖细胞中55 kDa的高尔基体重组堆积蛋白(GRASP55,由Gorasp2编码)相互作用。 Gorasp2-/-小鼠的产生和研究表明,基因敲除小鼠患有精子发生缺陷。在Gorasp2-/-小鼠中,精子中顶体的形成和JAM-C的极化定位发生了改变。另外,在Gorasp2-/-小鼠中,精子细胞的高尔基体形态受到干扰。与JAM-C或JAM-B配合使用的GRASP55的晶体结构表明,GRASP55通过PDZ介导的与JAM的相互作用而相互作用,并导致GRASP55的构象变化。一种计算机上药效团方法鉴定出一种名为Graspin的化合物,该化合物可抑制PDZ介导的GRASP55与JAM的相互作用。用Graspin处理小鼠会阻碍精子细胞中JAM-C的极化定位,诱导精子的过早释放,并影响减数分裂精细胞的高尔基形态。
  • 【淋巴管浸润在C型浸润性宫颈内膜腺癌中的作用。】 复制标题 收藏 收藏
    DOI:10.1097/PAS.0000000000000822 复制DOI
    作者列表:Roma AA,Park KJ,Xie H,De Vivar AD,Alvarado-Cabrero I,Rutgers JKL,Barbuto D,Silva EG
    BACKGROUND & AIMS: :Lymphovascular invasion (LVI) has been reported as an independent predictor of patient outcome in cervical carcinoma. However, not all studies support independent significance, especially in multivariable analyses. A risk stratification system recently introduced for endocervical adenocarcinoma was reported to better predict risk of lymph node (LN) metastasis. A subset of patients with tumors with pattern C features had LN metastasis and died of disease. In this study, we determined whether LVI had any additional significance in this subset of tumors. A total of 127 patients with pattern C tumors and at least 12-month follow-up were included. Tumors were separated into 3 subgroups. Those with no LVI and negative LNs represented 41 cases; most patients (36, 88%) were alive with no evidence of disease at last follow-up, whereas 4 (10%) died of disease, all after tumor recurrence/metastasis. Tumors with LVI, but negative LNs, represented 55 cases; recurrences were seen in 10 (18%) patients, of which 5 (50%) of them died of disease; remaining 5 patients are alive with persistent disease. Tumors with both LVI and positive LNs represented 31 cases; recurrences were seen in 13 (42%) patients; 11 (85%) patients died of disease and 2 are alive with persistent disease. One additional patient who presented with advanced stage also died of disease. Tumor size, horizontal spread, and LN status were significantly associated with outcome in univariate, but not in multivariable analysis; depth of invasion was not a predictor of outcome. Tumors with no LVI and negative LNs behaved significantly less aggressively than tumors with both LVI and positive LNs (P<0.01). LVI status (independent of LN status) was not significantly associated with patient outcome, although approached significance (P=0.06). In conclusion, LVI is a prerequisite for LN metastasis; however, by itself is not sufficient to predict tumor aggressiveness, whereas over 50% of patients with positive LNs died of disease. Stratifying pattern C tumors into subgroups based on LVI and LN status could further determine treatment in patients with pattern C tumors.
    背景与目标: :淋巴管浸润(LVI)被报告为宫颈癌患者预后的独立预测因子。但是,并非所有研究都支持独立意义,尤其是在多变量分析中。据报道,最近针对宫颈内膜腺癌引入的风险分层系统可以更好地预测淋巴结转移的风险。具有C型特征的部分肿瘤患者发生LN转移并死于疾病。在这项研究中,我们确定LVI在这部分肿瘤中是否还有其他意义。总共包括127位患有C型肿瘤且至少随访12个月的患者。将肿瘤分为3个亚组。没有LVI和LN阴性的患者代表41例;大多数患者(36%,88%)在最后一次随访时还活着,没有疾病的迹象,而4名(10%)死于疾病,都是在肿瘤复发/转移之后。 LVI阴性但LN阴性的肿瘤代表55例。 10例(18%)患者复发,其中5例(50%)死于疾病;其余5例患者仍患有持续性疾病。 LVI和LN阳性的肿瘤共占31例。 13例(42%)患者复发。 11名(85%)患者死于疾病,另有2例患有持续性疾病。另一位晚期患者也死于疾病。在单变量中,肿瘤大小,水平扩散和LN状态与预后显着相关,而在多变量分析中则无相关性。浸润深度不是预后的指标。没有LVI和LN阴性的肿瘤的侵袭性明显低于同时存在LVI和LN阳性的肿瘤(P <0.01)。 LVI状态(独立于LN状态)与患者预后没有显着相关性,尽管已接近显着性(P = 0.06)。总之,LVI是LN转移的先决条件。然而,仅靠其自身不足以预测肿瘤的侵袭性,而超过50%的LN阳性患者死于疾病。根据LVI和LN状况将C型肿瘤分为亚组可以进一步确定C型肿瘤患者的治疗方法。
  • 【发现双p38αN- {4- [5-(4-氟苯基)-3-甲基-2-甲基硫烷基-3H-咪唑-4-基]-吡啶-2-基}-乙酰胺(CBS-3595)具有抗TNFα相关疾病活性的MAPK / PDE-4抑制剂。】 复制标题 收藏 收藏
    DOI:10.1021/acs.jmedchem.6b01647 复制DOI
    作者列表:Albrecht W,Unger A,Bauer SM,Laufer SA
    BACKGROUND & AIMS: :The anti-inflammatory potential of p38 mitogen-activated protein kinase (MAPK) inhibitors was coincidentally expanded to a dual inhibition of p38α MAPK and phosphodiesterase 4 (PDE4), and the potential benefits arising from the blockage of both inflammation-related enzymes were thoroughly investigated. The most promising compound, CBS-3595 (1), was successively evaluated in in vitro experiments as well as in ex vivo and in vivo preclinical studies after administration of 1 to rodents, dogs, and monkeys. The resulting data clearly indicated a potent suppression of tumor necrosis factor alpha release. For reconfirming the findings of the animal studies when administering 1 to healthy human volunteers, a phase I clinical trial was conducted. Apart from further information regarding the pharmacokinetic and pharmacodynamic characteristics of 1, it was demonstrated that dual inhibition of p38α MAPK and PDE4 is able to synergistically attenuate the excessive anti-inflammatory response.
    背景与目标: :p38丝裂原活化蛋白激酶(MAPK)抑制剂的抗炎潜能同时扩展到了p38αMAPK和磷酸二酯酶4(PDE4)的双重抑制作用,并且两种炎症相关酶均被阻断产生了潜在的益处调查。给啮齿动物,狗和猴子施用1后,在体外实验以及离体和体内临床前研究中相继评估了最有前途的化合物CBS-3595(1)。所得数据清楚地表明有效抑制了肿瘤坏死因子α的释放。为了确认对健康的人类志愿者给药1时动物研究的结果,进行了I期临床试验。除了有关1的药代动力学和药效学特性的进一步信息外,还证明了对p38αMAPK和PDE4的双重抑制能够协同减弱过度的抗炎反应。
  • 【整个哺乳动物NEDD8连接酶家族中独特的N末端乙酰化依赖性相互作用的结构保守性。】 复制标题 收藏 收藏
    DOI:10.1016/j.str.2012.10.013 复制DOI
    作者列表:Monda JK,Scott DC,Miller DJ,Lydeard J,King D,Harper JW,Bennett EJ,Schulman BA
    BACKGROUND & AIMS: :Little is known about molecular recognition of acetylated N termini, despite prevalence of this modification among eukaryotic cytosolic proteins. We report that the family of human DCN-like (DCNL) co-E3s, which promote ligation of the ubiquitin-like protein NEDD8 to cullin targets, recognizes acetylated N termini of the E2 enzymes UBC12 and UBE2F. Systematic biochemical and biophysical analyses reveal 40- and 10-fold variations in affinities among different DCNL-cullin and DCNL-E2 complexes, contributing to varying efficiencies of different NEDD8 ligation cascades. Structures of DCNL2 and DCNL3 complexes with N-terminally acetylated peptides from UBC12 and UBE2F illuminate a common mechanism by which DCNL proteins recognize N-terminally acetylated E2s and how selectivity for interactions dependent on N-acetyl-methionine are established through side chains recognizing distal residues. Distinct preferences of UBC12 and UBE2F peptides for inhibiting different DCNLs, including the oncogenic DCNL1 protein, suggest it may be possible to develop small molecules blocking specific N-acetyl-methionine-dependent protein interactions.
    背景与目标: :尽管在真核细胞溶质蛋白中普遍存在这种修饰,但对乙酰化N末端的分子识别知之甚少。我们报告说,人类DCN样(DCNL)co-E3s的家族,促进泛素样蛋白NEDD8与cullin目标的连接,识别E2酶UBC12和UBE2F的乙酰化N末端。系统的生化和生物物理分析表明,不同的DCNL-cullin和DCNL-E2复合物之间的亲和力变化分别为40倍和10倍,从而导致了不同NEDD8连接级联反应效率的变化。具有来自UBC12和UBE2F的N末端乙酰化肽的DCNL2和DCNL3配合物的结构阐明了DCNL蛋白质识别N末端乙酰化E2的共同机制以及如何通过识别远端残基的侧链建立依赖N-乙酰甲硫氨酸的相互作用的选择性。 UBC12和UBE2F肽对于抑制不同的DCNL(包括致癌DCNL1蛋白)的不同偏好,表明可能开发出阻断特定N-乙酰基-蛋氨酸依赖性蛋白相互作用的小分子。
  • 【盘尾丝虫和A虫中的腐胺N-乙酰基转移酶,一种酶,参与多胺降解和N-乙酰基腐胺的释放。】 复制标题 收藏 收藏
    DOI:10.1016/0166-6851(90)90199-v 复制DOI
    作者列表:Wittich RM,Walter RD
    BACKGROUND & AIMS: :A novel type of N-acetyltransferase, clearly different from the nuclear and cytosolic polyamine N-acetyltransferases of mammals, was recently found in the intestinal nematode Ascaris suum. The occurrence of this putrescine N-acetylating enzyme has also been noted in the filarial parasite Onchocerca volvulus. The enzyme was partially purified from adults of O. volvulus and A. suum by chromatography on DEAE-cellulose and cadaverine-Sepharose. Substrate specificities of the filarial enzyme resemble those of the N-acetyltransferase from A. suum, with respect to its preference for putrescine and other diamines above polyamines and histones. Additionally, both nematode enzymes acetylated histamine, whereas dopamine and serotonin were not accepted as substrates. The activities of the N-acetyltransferase from O. volvulus and A. suum were potently inhibited by the drug berenil; the type of inhibition was competitive with respect to putrescine. The inhibition constants for berenil were determined as 4.2 and 1.2 microM for the enzymes of O. volvulus and A. suum, the Km values for putrescine were found to be 330 microM and 250 microM, respectively. Putrescine N-acetyltransferase is discussed as a regulatory step in the degradation of excessive polyamines via polyamine oxidase to putrescine. At this branching point, putrescine is retained in the cell for de novo synthesis of spermidine and spermine, catabolized via diamine oxidase or acetylated to a suitable transport product for excretion.
    背景与目标: :最近在肠道线虫A虫中发现了一种新型的N-乙酰基转移酶,其明显不同于哺乳动物的核和胞质多胺N-乙酰基转移酶。还已经在丝状寄生虫Onchocerca volvulus中发现了这种腐胺N-乙酰化酶的存在。通过在DEAE-纤维素和尸胺-Sepharose上进行色谱分离,从肠和成虫的成虫中部分纯化该酶。就其对腐胺和其他多胺(高于多胺和组蛋白)的偏好而言,丝状酶的底物特异性类似于来自A. suum的N-乙酰基转移酶。另外,两种线虫酶均使组胺乙酰化,而多巴胺和5-羟色胺不被接受为底物。苯丙胺类药物有效地抑制了来自贪食弧菌和su。suum的N-乙酰基转移酶的活性。抑制类型相对于腐胺具有竞争性。测定苯乙胺醇对肠球菌和猪链球菌的酶的抑制常数为4.2和1.2μM,发现腐胺的Km值分别为330μM和250μM。腐胺N-乙酰基转移酶被讨论为通过聚胺氧化酶将过量的聚胺降解为腐胺的调节步骤。在该分支点,腐胺保留在细胞中,用于从头合成亚精胺和亚精胺,通过二胺氧化酶分解代谢或乙酰化为合适的转运产物以排泄。
  • 【TGF-β/ miR-155 / c-Ski的机制调节人冠状动脉内皮细胞的内皮-间质转化。】 复制标题 收藏 收藏
    DOI:10.1042/BSR20160603 复制DOI
    作者列表:Wang J,He W,Xu X,Guo L,Zhang Y,Han S,Shen D
    BACKGROUND & AIMS: :Human coronary artery endothelial cells (HCAECs) have the potential to undergo fibrogenic endothelial-mesenchymal transition (EndMT), which results in matrix-producing fibroblasts and thereby contributes to the pathogenesis of cardiac fibrosis. Recently, the profibrotic cytokine transforming growth factor-β (TGF-β) is shown to be the crucial pathogenic driver which has been verified to induce EndMT. C-Ski is an important regulator of TGF-β signaling. However, the detailed role of c-Ski and the molecular mechanisms by which c-Ski affects TGF-β-induced EndMT in HCAECs are not largely elucidated. In the present study, we treated HCAECs with TGF-β of different concentrations to induce EndMT. We found that overexpression of c-Ski in HCAECs either blocked EndMT via hindering Vimentin, Snail, Slug, and Twist expression while enhancing CD31 expression, with or without TGF-β treatment. In contrast, suppression of c-Ski further enhanced EndMT. Currently, miRNA expression disorder has been frequently reported associating with cardiac fibrosis. By using online tools, we regarded miR-155 as a candidate miRNA that could target c-Ski, which was verified using luciferase assays. C-Ski expression was negatively regulated by miR-155. TGF-β-induced EndMT was inhibited by miR-155 silence; the effect of TGF-β on Vimentin, CD31, Snail, Slug, and Twist could be partially restored by miR-155. Altogether, these findings will shed light on the role and mechanism by which miR-155 regulates TGF-β-induced HCAECs EndMT via c-Ski to affect cardiac fibrosis, and miR-155/c-Ski may represent novel biomarkers and therapeutic targets in the treatment of cardiac fibrosis.
    背景与目标: :人冠状动脉内皮细胞(HCAEC)有可能经历纤维化内皮-间充质转变(EndMT),这会导致产生基质的成纤维细胞,从而促进心脏纤维化的发病机理。最近,已证明原纤维化细胞因子转化生长因子-β(TGF-β)是关键的致病性驱动因子,已被证实可诱导EndMT。 C-Ski是TGF-β信号传导的重要调节剂。然而,在很大程度上没有阐明c-Ski的详细作用以及c-Ski影响HCAECs中TGF-β诱导的EndMT的分子机制。在本研究中,我们用不同浓度的TGF-β处理HCAEC,以诱导EndMT。我们发现,无论是否使用TGF-β处理,HCAEC中c-Ski的过表达要么通过阻碍波形蛋白,蜗牛,Slug和Twist的表达而阻断EndMT,同时增强CD31的表达。相反,抑制c-Ski进一步增强了EndMT。当前,miRNA表达障碍经常被报道与心脏纤维化有关。通过使用在线工具,我们将miR-155视为可以靶向c-Ski的候选miRNA,这已通过荧光素酶测定法进行了验证。 C-Ski表达受到miR-155的负调控。 TGF-β诱导的EndMT被miR-155沉默抑制; TGF-β对波形蛋白,CD31,蜗牛,Slug和Twist的作用可以被miR-155部分恢复。总而言之,这些发现将阐明miR-155通过c-Ski调节TGF-β诱导的HCAECs EndMT的作用和机制,从而影响心脏纤维化,而miR-155 / c-Ski可能代表了新型的生物标志物和治疗靶点。心脏纤维化的治疗。

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