• 【应激特异性p38 MAPK激活足以驱动EGFR内吞作用,但不足以驱动其核易位。】 复制标题 收藏 收藏
    DOI:10.1242/jcs.202358 复制DOI
    作者列表:Tomas A,Jones S,Vaughan SO,Hochhauser D,Futter CE
    BACKGROUND & AIMS: :EGF receptor (EGFR) endocytosis is induced by stress in a manner dependent on the p38 MAPK family. Ligand and stresses such as X-rays, reportedly promote nuclear trafficking of endocytosed EGFR for regulation of gene transcription and DNA repair. We fail to detect EGFR endocytosis or nuclear transport following X-ray treatment of HeLa or head and neck cancer cells, despite extensive DNA damage induction. Apparent nuclear staining with EGFR extracellular domain antibody remained present despite reduced/absent EGFR expression, and so did not represent nuclear EGFR. UVB and UVC, but not X-ray or UVA, treatment induced p38 activation and EGFR endocytosis, although all of these stresses induced DNA damage, indicating that DNA damage alone is not sufficient to induce EGFR endocytosis. Increased reactive oxygen species (ROS) levels following UVB treatment, compared to that seen with X-rays, do not alone explain differences in p38 activation. UVB, like UVC, induced EGFR accumulation predominantly in perinuclear endosomes, rather than in the nucleus. Our morphological techniques identifying major changes in receptor distribution do not exclude the possibility that small but biologically relevant amounts of EGFR enter the nucleus. This study highlights the importance and limitations of morphological analyses of receptor distribution in understanding signaling outcome.
    背景与目标: :EGF受体(EGFR)的内吞作用是由应激以依赖于p38 MAPK家族的方式诱导的。据报道,配体和压力(例如X射线)可促进内吞EGFR的核转运,从而调节基因转录和DNA修复。尽管广泛的DNA损伤诱导作用,但在X射线治疗HeLa或头颈部癌细胞后,我们仍未检测到EGFR的内吞作用或核转运。尽管EGFR表达减少/缺失,但仍存在EGFR胞外域抗体的明显核染色,因此并不代表核EGFR。 UVB和UVC处理可诱导p38活化和EGFR内吞作用,而不是X射线或UVA,尽管所有这些压力均可诱导DNA损伤,表明仅DNA损伤不足以诱导EGFR内吞作用。与X射线相比,UVB处理后增加的活性氧(ROS)水平不能单独解释p38活化的差异。与UVC一样,UVB主要诱导EGFR聚集在核周内体中,而不是在细胞核中。我们的鉴定受体分布主要变化的形态学技术并不排除少量但生物学上相关的EGFR进入细胞核的可能性。这项研究强调了受体分布形态分析在理解信号转导结果中的重要性和局限性。
  • 【针对携带罕见EGFR突变的非小细胞肺癌的第一代EGFR-TKIs的结果:BE阳性研究的事后分析。】 复制标题 收藏 收藏
    DOI:10.1016/j.cllc.2017.05.016 复制DOI
    作者列表:
    BACKGROUND & AIMS: BACKGROUND:Beyond progression after tyrosine kinase inhibitor in EGFR-positive non-small-cell lung cancer patients (BE-POSITIVE) was the first Italian multicenter observational study that reported the outcomes of first-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in a "real-life" Caucasian EGFR-mutated non-small-cell lung cancer (NSCLC) population. The sharing of multi-institutional experiences represents a crucial strategy to enrich knowledge about uncommon EGFR mutations. Therefore, we performed a post hoc analysis of the BE-POSITIVE study. PATIENTS AND METHODS:Data of advanced NSCLC patients with uncommon EGFR mutations who received first-line first-generation EGFR-TKIs in 24 Italian Hospitals were collected. In this analysis we aimed to evaluate overall survival (OS), progression-free survival (PFS), and overall response rate (ORR) of EGFR-TKIs in NSCLC patients harboring uncommon EGFR mutations. RESULTS:Thirty-five patients harboring uncommon EGFR mutations (any mutation other than deletion 19 or substitution of leucine by arginine at codon 858) were included of the original 312 EGFR-mutated cases. Most of them were female (n = 20, 57.1%), former smokers (n = 23, 65.7%), with adenocarcinoma (n = 31, 88.6%). The most frequent EGFR mutations were G719X (n = 6, 17.2%) and L861Q (n = 5, 14.2%). The population presented an ORR of 25.7%, a median PFS of 5.19 months, and a median OS of 14.49 months. When stratified according to type of EGFR mutation, median OS ranged from 3.65 months for unspecified mutations to 21.29 for double EGFR mutations. Median PFS ranged from 1.77 months for unspecified mutations to 20.83 months for concomitant EGFR-anaplastic lymphoma kinase alteration. ORR varied from 0% in exon 18, 20 and double gene alteration to 66.6% in exon 19. CONCLUSION:Our study supports the existence of a strong outcome heterogeneity within patients harboring uncommon EGFR mutations, which needs to be clarified to achieve a real personalized treatment strategy.
    背景与目标: 背景:酪氨酸激酶抑制剂在EGFR阳性非小细胞肺癌患者中取得的突破性进展(BE-POSITIVE)是第一项意大利多中心观察性研究,该研究报告了第一代表皮生长因子受体(EGFR)-酪氨酸激酶的结果现实生活中的白种人EGFR突变的非小细胞肺癌(NSCLC)人群中存在多种抑制剂(TKIs)。共享多机构经验代表了一项重要策略,可以丰富有关罕见的EGFR突变的知识。因此,我们对正研究进行了事后分析。
    患者和方法:收集了意大利24所医院接受一线第一代EGFR-TKI的EGFR突变罕见的晚期NSCLC患者的数据。在这项分析中,我们旨在评估具有罕见EGFR突变的NSCLC患者的EGFR-TKIs的总生存期(OS),无进展生存期(PFS)和总缓解率(ORR)。
    结果:35例罕见的EGFR突变病例包括了不常见的EGFR突变(除19号缺失或亮氨酸在858位密码子处亮氨酸替代以外的任何突变)。其中大多数是女性(n = 20,57.1%),曾吸烟者(n = 23,65.7%),患有腺癌(n = 31,88.6%)。最常见的EGFR突变是G719X(n = 6,17.2%)和L861Q(n = 5,14.2%)。人群的ORR为25.7%,PFS中位数为5.19个月,OS中位数为14.49个月。根据EGFR突变的类型进行分层时,中位OS​​范围从3.65个月(未指定突变)到21.29倍(双重EGFR突变)不等。 PFS的中位值范围从未指定突变的1.77个月到伴随的EGFR-再生障碍性淋巴瘤激酶改变的20.83个月不等。 ORR从外显子18、20和双基因改变的0%到外显子19的66.6%不等。
    结论:我们的研究支持携带罕见EGFR突变的患者存在强大的预后异质性,需要阐明这一点以实现真正的个性化治疗策略。
  • 【IGF信号基因的复发突变和骨肉瘤中基因组重排的不同模式。】 复制标题 收藏 收藏
    DOI:10.1038/ncomms15936 复制DOI
    作者列表:
    BACKGROUND & AIMS: :Osteosarcoma is a primary malignancy of bone that affects children and adults. Here, we present the largest sequencing study of osteosarcoma to date, comprising 112 childhood and adult tumours encompassing all major histological subtypes. A key finding of our study is the identification of mutations in insulin-like growth factor (IGF) signalling genes in 8/112 (7%) of cases. We validate this observation using fluorescence in situ hybridization (FISH) in an additional 87 osteosarcomas, with IGF1 receptor (IGF1R) amplification observed in 14% of tumours. These findings may inform patient selection in future trials of IGF1R inhibitors in osteosarcoma. Analysing patterns of mutation, we identify distinct rearrangement profiles including a process characterized by chromothripsis and amplification. This process operates recurrently at discrete genomic regions and generates driver mutations. It may represent an age-independent mutational mechanism that contributes to the development of osteosarcoma in children and adults alike.
    背景与目标: :骨肉瘤是影响儿童和成人的原发性骨恶性肿瘤。在这里,我们介绍了迄今为止最大的骨肉瘤测序研究,包括112种儿童和成人肿瘤,涵盖了所有主要的组织学亚型。我们研究的关键发现是在8/112(7%)的病例中鉴定胰岛素样生长因子(IGF)信号基因的突变。我们在另外87个骨肉瘤中使用荧光原位杂交(FISH)验证了这一观察结果,并在14%的肿瘤中观察到了IGF1受体(IGF1R)扩增。这些发现可能会为将来在骨肉瘤中使用IGF1R抑制剂的患者选择药物提供依据。分析突变的模式,我们确定了独特的重排图谱,包括以色杆菌病和扩增为特征的过程。该过程在离散的基因组区域反复进行,并产生驱动突变。它可能代表了与年龄无关的突变机制,有助于儿童和成年人中骨肉瘤的发展。
  • 【定量共表达EGFR和HER2对HER激活和运输的影响。】 复制标题 收藏 收藏
    DOI:10.1016/j.bbrc.2008.04.043 复制DOI
    作者列表:Shankaran H,Zhang Y,Opresko L,Resat H
    BACKGROUND & AIMS: :The human epidermal growth factor receptor (HER) system is an intricately regulated system that plays critical roles in development and tumorigenesis. Here, we apply integrated experimentation and modeling to analyze HER receptor activation in a panel of cell lines expressing endogenous levels of EGFR/HER1 and different levels of HER2. A mathematical model that includes the fundamental processes involved in receptor activation and trafficking was used to fit the experimental data, and values of the independent parameters for active receptor dimer formation affinities, trafficking rates and relative phosphorylation levels were estimated. Obtained parameter values quantitatively support the existing ideas on the effect of HER2 on EGFR dynamics, and enable us to predict the abundances of various phosphorylated receptor dimers in the cell lines.
    背景与目标: :人类表皮生长因子受体(HER)系统是一个复杂的调控系统,在发育和肿瘤发生中起着至关重要的作用。在这里,我们应用综合实验和建模来分析一组表达内源性EGFR / HER1和不同水平HER2的细胞系中的HER受体激活。使用包括参与受体激活和运输的基本过程的数学模型来拟合实验数据,并估计活性受体二聚体形成亲和力,运输速率和相对磷酸化水平的独立参数的值。获得的参数值定量支持关于HER2对EGFR动力学影响的现有观点,并使我们能够预测细胞系中各种磷酸化受体二聚体的丰度。
  • 【携带TP63基因从头突变的患者的犁骨发育不良(3q27)。】 复制标题 收藏 收藏
    DOI:10.1016/j.ijporl.2013.06.027 复制DOI
    作者列表:Schindler A,Guazzarotti L,Mameli C,Urbani E,Mozzanica F,Guerrini L,Zuccotti GV
    BACKGROUND & AIMS: :The congenital vomer defect (CVD) is a rare and still partially unknown condition. Only few cases have been reported in the international literature and the large majority of them appeared to be isolated. We report a case of CVD detected in a 7-year-old girl affected by ectodermal dysplasia clefting syndrome caused by a mutation of the TP63 gene.
    背景与目标: :先天性犁骨缺损(CVD)是一种罕见且仍部分未知的疾病。国际文献中仅报道了少数病例,其中大多数似乎是孤立的。我们报告了一个在7岁的女孩中检测到的CVD病例,该女孩受TP63基因突变引起的外胚层发育不良裂口综合征的影响。
  • 【线粒体TIM10亚基中保守带电残基的突变排除了TIM10复合物的组装,但并未消除酵母细胞的生长。】 复制标题 收藏 收藏
    DOI:10.1016/j.jmb.2007.06.025 复制DOI
    作者列表:Vergnolle MA,Alcock FH,Petrakis N,Tokatlidis K
    BACKGROUND & AIMS: :The Saccharomyces cerevisiae TIM10 complex (TIM10c) is an ATP-independent chaperone of the mitochondrial intermembrane space, involved in transport of polytopic membrane proteins. The complex is an alpha(3)beta(3) hexamer of Tim9 and Tim10 subunits. We have generated specific mutations in charged residues in the central core domain of each subunit delineated by the characteristic twin CX(3)C motif, and investigated the effect of these mutations on subunit folding, complex assembly and TIM10 function in vitro and in vivo. Any combination of mutations that included a specific glutamate residue, conserved in all known Tim9 and Tim10 sequences, abolished assembly of the TIM10 complex. In vivo complementation analyses using a MET3-TIM10 strain that is selectively inactivated for the expression of wild-type Tim10 showed that (i) an N-terminal deleted version of Tim10 that was previously shown to be defective in substrate binding is lethal under all conditions, but (ii) the charged residues mutant of Tim10 that is defective in assembly with Tim9 can restore growth in glucose, but not in non-fermentable carbon sources. These data suggest that formation of the hexamer is beneficial but not vital for TIM10 function, whilst the N-terminal substrate-binding region of Tim10 is essential in vivo.
    背景与目标: :酿酒酵母TIM10复合物(TIM10c)是线粒体膜间空间的一个不依赖ATP的分子伴侣,参与多膜蛋白的转运。该复合物是Tim9和Tim10亚基的alpha(3)beta(3)六聚体。我们已经在每个亚基的中央核心域带电残基中生成特定的突变,这些突变由特征性双胞胎CX(3)C图案描绘,并研究了这些突变对亚基折叠,复杂装配和TIM10功能的影响。在所有已知的Tim9和Tim10序列中均保守的,包括特定谷氨酸残基的任何突变组合都可消除TIM10复合体的装配。使用针对野生型Tim10的表达而选择性失活的MET3-TIM10菌株的体内互补分析显示(i)先前被证明在底物结合方面有缺陷的Tim10的N末端缺失版本在所有情况下都是致死性的,但(ii)与Tim9组装有缺陷的Tim10的带电荷残基突变体可以恢复葡萄糖的生长,但不能恢复不可发酵的碳源。这些数据表明六聚体的形成对TIM10功能是有益的,但不是至关重要的,而Tim10的N端底物结合区在体内则是必不可少的。
  • 【ErbB3,EGFR和Erk的激活对于人类乳腺癌细胞株具有对氟维司群的抗性至关重要。】 复制标题 收藏 收藏
    DOI:10.1007/s10549-008-0011-8 复制DOI
    作者列表:Frogne T,Benjaminsen RV,Sonne-Hansen K,Sorensen BS,Nexo E,Laenkholm AV,Rasmussen LM,Riese DJ 2nd,de Cremoux P,Stenvang J,Lykkesfeldt AE
    BACKGROUND & AIMS: :Seven fulvestrant resistant cell lines derived from the estrogen receptor alpha positive MCF-7 human breast cancer cell line were used to investigate the importance of epidermal growth factor receptor (ErbB1-4) signaling. We found an increase in mRNA expression of EGFR and the ErbB3/ErbB4 ligand heregulin2 (hrg2) and a decrease of ErbB4 in all resistant cell lines. Western analyses confirmed the upregulation of EGFR and hrg2 and the downregulation of ErbB4. Elevated activation of EGFR and ErbB3 was seen in all resistant cell lines and the ErbB3 activation occurred by an autocrine mechanism. ErbB4 activation was observed only in the parental MCF-7 cells. The downstream kinases pAkt and pErk were increased in five of seven and in all seven resistant cell lines, respectively. Treatment with the EGFR inhibitor gefitinib preferentially inhibited growth and reduced the S phase fraction in the resistant cell lines concomitant with inhibition of Erk and unaltered Akt activation. In concert, inhibition of Erk with U0126 preferentially reduced growth of resistant cell lines. Treatment with ErbB3 neutralizing antibodies inhibited ErbB3 activation and resulted in a modest but statistically significant growth inhibition of two resistant cell lines. These data indicate that ligand activated ErbB3 and EGFR, and Erk signaling play important roles in fulvestrant resistant cell growth. Furthermore, the decreased level of ErbB4 in resistant cells may facilitate heterodimerization of ErbB3 with EGFR and ErbB2. Our data support that a concerted action against EGFR, ErbB2 and ErbB3 may be required to obtain complete growth suppression of fulvestrant resistant cells.
    背景与目标: :使用七种来自雌激素受体α阳性MCF-7人乳腺癌细胞的抗氟维司群抗性细胞系,调查表皮生长因子受体(ErbB1-4)信号传导的重要性。我们发现在所有耐药细胞系中EGFR和ErbB3 / ErbB4配体heregulin2(hrg2)的mRNA表达增加,而ErbB4的减少。 Western分析证实了EGFR和hrg2的上调以及ErbB4的下调。在所有耐药细胞系中均观察到EGFR和ErbB3的激活增强,并且ErbB3的激活是通过自分泌机制发生的。仅在亲本MCF-7细胞中观察到ErbB4激活。下游激酶pAkt和pErk在七个抗性细胞系中的五个中和在所有七个抗性细胞系中分别增加。用EGFR抑制剂吉非替尼治疗可优先抑制生长并降低耐药细胞系中的S期分数,同时抑制Erk和未改变的Akt激活。一致地,用U0126抑制Erk优先降低了抗性细胞系的生长。用ErbB3中和抗体处理可抑制ErbB3活化,并导致对两种抗性细胞系的抑制作用达到中等但统计学上显着的增长。这些数据表明配体激活的ErbB3和EGFR,以及Erk信号传导在耐氟司韦特的细胞生长中起重要作用。此外,耐药细胞中ErbB4水平降低可能有助于ErbB3与EGFR和ErbB2异源二聚化。我们的数据支持可能需要针对EGFR,ErbB2和ErbB3的协同作用才能获得对氟维司群抗性细胞的完全生长抑制。
  • 【TP63中的新突变与年龄相关的病理有关。】 复制标题 收藏 收藏
    DOI:10.1038/sj.ejhg.5201888 复制DOI
    作者列表:Holder-Espinasse M,Martin-Coignard D,Escande F,Manouvrier-Hanu S
    BACKGROUND & AIMS: :Increases in the number of allelic malformation syndromes have led to their classification according to their pathogenesis rather than their clinical specific phenotype. TP63 (also known as TP73L) mutations have been identified in several such syndromes characterized by autosomal dominant transmission and various combinations of ectodermal dysplasia, limb malformations and orofacial clefting. TP63 has not yet been implicated in early aging phenotype in humans, even though p63 activates a program of cellular senescence and p63-compromised mice display features of accelerated aging. We report on a family with four affected adult females presenting with Rapp-Hodgkin syndrome (RHS), an autosomal dominant clinical entity that associates anhidrotic ectodermal dysplasia with cleft lip and palate. Features between RHS and EEC syndrome (ectrodactyly, ectodermal dysplasia and cleft lip/palate) have led to the recent identification of mutations in the TP63 gene, located on 3q27, in this condition. Our patients present typical clinical features of RHS, but also ophthalmic anomalies such as corneal dystrophy and premature menopause (around 30 years). The latter findings have never been reported in this condition, and could be secondary to a new TP63 deletion that has been identified in this family.
    背景与目标: :等位基因畸形综合征数量的增加已导致根据其发病机理而不是根据临床特定表型对它们进行分类。 TP63(也称为TP73L)突变已在几种以常染色体显性遗传和外胚层发育不良,肢体畸形和口唇裂的各种组合为特征的综合症中得到鉴定。 TP63尚未涉及人类的早期衰老表型,即使p63激活了细胞衰老程序并且p63受损的小鼠也表现出加速衰老的特征。我们报告了一个家庭,该家庭有四名受影响的成年女性,表现出拉普霍奇金综合征(RHS),这是一种常染色体显性临床实体,将无角质外胚层发育不良与唇left裂相关。 RHS和EEC综合征之间的特征(实际上是外胚层发育异常和唇裂/唇pal裂)导致这种情况下最近鉴定出位于3q27的TP63基因突变。我们的患者表现出RHS的典型临床特征,还表现出眼科异常,例如角膜营养不良和更年期提前(约30年)。后者的发现从未在这种情况下报道过,可能是该家族中已发现的新的TP63缺失的继发者。
  • 【在人支气管上皮细胞中,TGF-β1介导的COX-2诱导需要EGFR信号传导。】 复制标题 收藏 收藏
    DOI:10.1165/rcmb.2007-0100OC 复制DOI
    作者列表:Liu M,Yang SC,Sharma S,Luo J,Cui X,Peebles KA,Huang M,Sato M,Ramirez RD,Shay JW,Minna JD,Dubinett SM
    BACKGROUND & AIMS: :Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins and thromboxanes from free arachidonic acid. Increasing evidence suggests that COX-2 plays a role in tumorigenesis. A variety of stimuli induce COX-2 and it is overexpressed in many tumors, including non-small cell lung cancer (NSCLC). We studied the regulation of COX-2 expression in immortalized human bronchial epithelial cells (HBECs) by transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF) because these two growth factors are present in both the pulmonary milieu of those at risk for lung cancer as well as in the tumor microenvironment. EGF significantly enhanced TGF-beta1-mediated induction of COX-2 and corresponding prostaglandin E2 (PGE2) production. TGF-beta1 and EGF induced COX-2 at the transcriptional and post-transcriptional levels. EGF receptor (EGFR) inhibition, neutralizing antibody against amphiregulin, or mitogen-activated protein kinase kinase (MEK) inhibition blocked TGF-beta1-mediated COX-2 induction. COX-2 induction by TGF-beta1 depended upon Smad3 signaling and required the activity of EGFR or its downstream mediators. Autocrine amphiregulin signaling maintains EGFR in a constitutively active state in HBECs, allowing for COX-2 induction by TGF-beta1. Thus, EGFR ligands, which are abundant in the pulmonary microenvironment of those at risk for lung cancer, potentiate and are required for COX-2 induction by TGF-beta1 in HBEC. These findings emphasize the central role of EGFR signaling in COX-2 induction by TGF-beta1 and suggest that inhibition of EGFR signaling should be investigated further for lung cancer prevention.
    背景与目标: :环氧合酶2(COX-2)是由游离花生四烯酸生产前列腺素和血栓烷的关键酶。越来越多的证据表明,COX-2在肿瘤发生中起作用。多种刺激可诱导COX-2的表达,并在许多肿瘤中过度表达,包括非小细胞肺癌(NSCLC)。我们通过转化生长因子-beta1(TGF-beta1)和表皮生长因子(EGF)研究了永生化人支气管上皮细胞(HBECs)中COX-2表达的调节,因为这两个生长因子均存在于它们的肺环境中处于肺癌以及肿瘤微环境中的风险。 EGF显着增强了TGF-beta1介导的COX-2诱导和相应的前列腺素E2(PGE2)的产生。 TGF-beta1和EGF在转录和转录后水平诱导COX-2。 EGF受体(EGFR)抑制,抗双调蛋白的中和抗体或丝裂原激活的蛋白激酶激酶(MEK)抑制可阻止TGF-beta1介导的COX-2诱导。 TGF-beta1诱导COX-2依赖于Smad3信号传导,并需要EGFR或其下游介质的活性。自分泌两性调节蛋白信号转导使HBEC中的EGFR保持组成型活性状态,从而允许TGF-beta1诱导COX-2。因此,在有肺癌风险的人的肺微环境中丰富的EGFR配体增强了,并且是HBEC中TGF-beta1诱导COX-2所必需的。这些发现强调了EGFR信号传导在TGF-beta1诱导COX-2诱导中的核心作用,并建议应进一步研究EGFR信号传导的抑制作用以预防肺癌。
  • 【小麦胞质乙酰辅酶A羧化酶可补充酵母中的ACC1空突变。】 复制标题 收藏 收藏
    DOI:10.1073/pnas.94.18.9990 复制DOI
    作者列表:Joachimiak M,Tevzadze G,Podkowinski J,Haselkorn R,Gornicki P
    BACKGROUND & AIMS: :Spores harboring an ACC1 deletion derived from a diploid Saccharomyces cerevisiae strain, in which one copy of the entire ACC1 gene is replaced with a LEU2 cassette, fail to grow. A chimeric gene consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat cytosolic acetyl-CoA carboxylase (ACCase) cDNA, and yeast ACC1 3' tail was used to complement a yeast ACC1 mutation. The complementation demonstrates that active wheat ACCase can be produced in yeast. At low concentrations of galactose, the activity of the "wheat gene" driven by the GAL10 promoter is low and ACCase becomes limiting for growth, a condition expected to enhance transgenic yeast sensitivity to wheat ACCase-specific inhibitors. An aryloxyphenoxypropionate and two cyclohexanediones do not inhibit growth of haploid yeast strains containing the yeast ACC1 gene, but one cyclohexanedione inhibits growth of the gene-replacement strains at concentrations below 0.2 mM. In vitro, the activity of wheat cytosolic ACCase produced by the gene-replacement yeast strain is inhibited by haloxyfop and cethoxydim at concentrations above 0.02 mM. The activity of yeast ACCase is less affected. The wheat plastid ACCase in wheat germ extract is inhibited by all three herbicides at concentrations below 0.02 mM. Yeast gene-replacement strains will provide a convenient system for the study of plant ACCases.
    背景与目标: :带有源自二倍体酿酒酵母菌株的ACC1缺失的孢子无法生长,其中整个ACC1基因的一个拷贝被LEU2盒代替。由酵母GAL10启动子,酵母ACC1前导序列,小麦胞质乙酰辅酶A羧化酶(ACCase)cDNA和酵母ACC1 3'尾部组成的嵌合基因用于补充酵母ACC1突变。互补表明,可以在酵母中产生活性小麦ACCase。在低浓度的半乳糖下,由GAL10启动子驱动的“小麦基因”的活性低,ACCase成为限制生长的条件,该条件有望增强转基因酵母对小麦ACCase特异性抑制剂的敏感性。芳氧基苯氧基丙酸酯和两种环己二酮不抑制含有酵母ACC1基因的单倍体酵母菌株的生长,但是一种环己二酮在低于0.2 mM的浓度下抑制基因置换菌株的生长。在体外,由基因替代酵母菌株产生的小麦胞质ACCase的活性在0.02 mM以上的浓度下被haloxyfop和cethoxydim抑制。酵母ACCase的活性受到的影响较小。浓度低于0.02 mM的所有三种除草剂均能抑制小麦胚芽提取物中的小麦质体ACCase。酵母基因置换菌株将为植物ACCases的研究提供方便的系统。
  • 【域特定的模拟磷酸化突变允许解剖不同的蛋白激酶C(PKC)同型触发的RNA结合蛋白HuR的活动。】 复制标题 收藏 收藏
    DOI:10.1016/j.cellsig.2013.08.003 复制DOI
    作者列表:Schulz S,Doller A,Pendini NR,Wilce JA,Pfeilschifter J,Eberhardt W
    BACKGROUND & AIMS: :The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA.
    背景与目标: :普遍存在的mRNA结合蛋白人类抗原R(HuR)参与许多富含AU元素(ARE)的mRNA的转录后调控。以前,通过使用体外激酶测定,我们已经将丝氨酸(Ser)158、221和318确定为蛋白激酶C(PKC)触发的磷酸化的靶标。在这项研究中,我们测试了在不同的PKC目标位点上带有磷酸仿Ser(S)-Asp(D)取代的GFP标记或GST标记的HuR构建体是否会影响不同的HuR功能,包括HuR核质重分布和结合对不同类型的含有ARE的mRNA的表达。与野生型HuR相比,在HuR的铰链区(HuR-S221D)带有磷酸模拟取代的磷酸化GFP标记的HuR蛋白显示出增加的细胞质丰度。相反,来自体外激酶测定和电泳迁移率变动测定(EMSA)的数据暗示Ser 221的磷酸化与HuR的mRNA结合无关。定量标记GST标签的野生型HuR和相应的在磷酸RRM2(HuR-S158D)或RRM3(HuR-S318D)中具有磷酸模拟取代作用的HuR蛋白的体外结合亲和力表明通过微型热电泳(MST) -在高纳摩尔范围内将HuR型转变为I,II或III型-ARE-寡核苷酸。有趣的是,位置158或318处的磷酸模拟突变对HuR与I型和II型ARE-mRNA的结合具有负面影响,而它显着增强了HuR对III型ARE底物的亲和力。我们的数据表明,PKCs在不同的HuR域上对HuR的磷酸化差异会协调亚细胞HuR的分布,并导致与富含U的靶标mRNA的优先结合。
  • 【在智障,早期发作性痉挛和小脑萎缩的家庭中,外显子组测序可检测出EXOSC3中的新突变。】 复制标题 收藏 收藏
    DOI:10.1007/s10048-013-0371-z 复制DOI
    作者列表:Zanni G,Scotton C,Passarelli C,Fang M,Barresi S,Dallapiccola B,Wu B,Gualandi F,Ferlini A,Bertini E,Wei W
    BACKGROUND & AIMS: :Whole exome sequencing in two-generational kindred from Bangladesh with early onset spasticity, mild intellectual disability, distal amyotrophy, and cerebellar atrophy transmitted as an autosomal recessive trait identified the following two missense mutations in the EXOSC3 gene: a novel p.V80F mutation and a known p.D132A change previously associated with mild variants of pontocerebellar hypoplasia type 1. This study confirms the involvement of RNA processing proteins in disorders with motor neuron and cerebellar degeneration overlapping with spinocerebellar ataxia 36 and rare forms of hereditary spastic paraplegia with cerebellar features.
    背景与目标: :来自孟加拉国的两代亲属的全外显子组测序,具有发作前的痉挛,轻度智力残疾,远端肌萎缩和小脑萎缩(作为常染色体隐性遗传),它们确定了EXOSC3基因中的以下两个错义突变:一种新的p.V80F突变和一种已知的p.D132A改变,先前与1型小脑小脑发育不全的轻度变异有关。这项研究证实了RNA加工蛋白参与了运动神经元和小脑变性疾病与脊髓小脑性共济失调36和罕见的具有小脑特征的遗传性痉挛性截瘫的疾病。
  • 【表皮生长因子受体(EGFR)抑制剂和衍生疗法。】 复制标题 收藏 收藏
    DOI:10.1093/annonc/mds351 复制DOI
    作者列表:Dziadziuszko R,Jassem J
    BACKGROUND & AIMS: :Epidermal growth factor receptor inhibitors are used to treat advanced lung cancer patients for almost a decade. Current knowledge on their role in the first or subsequent lines of therapy serves as a model for other targeted therapies in development. Several molecular predictors of outcomes were successfully identified in preclinical and clinical studies. Evaluation of EGFR-activating mutations is currently used to define biologically distinct patient subsets with important consequences for prognosis and therapy. Ongoing translational and clinical research exploring EGFR inhibition in lung cancer focuses on better understanding of biology of EGFR-driven disease, efficacy of novel irreversible EGFR inhibitors and monoclonal antibodies, efficacy of combination strategies, and attempts to move EGFR inhibitors into therapy portfolio for early-stage disease.
    背景与目标: 表皮生长因子受体抑制剂被用于治疗晚期肺癌患者近十年。目前关于它们在治疗的第一线或后续线中的作用的知识可作为开发中其他靶向疗法的模型。在临床前和临床研究中成功鉴定了几种预后的分子预测因子。 EGFR激活突变的评估目前用于定义生物学上不同的患者亚组,对预后和治疗产生重要影响。正在进行的针对肺癌中EGFR抑制的转化和临床研究致力于更好地理解EGFR驱动疾病的生物学特性,新型不可逆EGFR抑制剂和单克隆抗体的功效,联合策略的功效以及尝试将EGFR抑制剂纳入早期治疗方案中分期疾病。
  • 【由于新的Gln281-> Arg突变,导致血浆血小板活化因子乙酰水解酶的活性降低。】 复制标题 收藏 收藏
    DOI:10.1006/bbrc.1997.7047 复制DOI
    作者列表:Yamada Y,Yokota M
    BACKGROUND & AIMS: The prevalence of plasma platelet-activating factor (PAF) acetylhydrolase deficiency was investigated in 477 healthy Japanese individuals and 985 patients with various cardiovascular diseases. The genotype for this enzyme with regard to a G994-->T mutation (MM, normal; Mm, heterozygote; mm, mutant homozygote) was determined by an allele-specific polymerase chain reaction in 80 subjects shown to have no or low plasma activity (<10 nmol/min/ml). In 72 subjects, the genotype was consistent with plasma enzyme activity; 44 individuals with no activity were mm, and 28 with low activity were Mm. However, eight subjects with the MM genotype showed plasma enzyme activities of <10 nmol/min/ml. Determination of the DNA sequence of exon 9 of the plasma PAF acetylhydrolase gene in these eight subjects revealed a previously unidentified A1001-->G missense mutation, resulting in a Gln281-->Arg substitution, in a 72-year-old woman with coronary artery disease, essential hypertension, and no plasma enzyme activity. Site-directed mutagenesis in vitro showed that the corresponding recombinant mutant protein lacked PAF acetylhydrolase activity. Thus, the Gln281-->Arg substitution appears responsible for the loss of plasma PAF acetylhydrolase activity.

    背景与目标: 在477名健康的日本人和985名患有各种心血管疾病的患者中调查了血浆血小板活化因子(PAF)乙酰水解酶缺乏症的患病率。通过等位基因特异性聚合酶链反应在80名无或血浆活性低的受试者中确定了与G994-> T突变有关的该酶的基因型(MM,正常; Mm,杂合子; mm,突变纯合子) (<10 nmol / min / ml)。在72名受试者中,基因型与血浆酶活性一致。 mm没有活动的个体为44,而Mm为活动的个体28。但是,有MM基因型的八名受试者显示血浆酶活性<10 nmol / min / ml。在这八名受试者中,血浆PAF乙酰水解酶基因外显子9的DNA序列测定表明,在72岁的冠状动脉女性中,先前未确定的A1001-> G错义突变,导致了Gln281-> Arg替代。动脉疾病,原发性高血压,且无血浆酶活性。体外定点诱变显示相应的重组突变蛋白缺乏PAF乙酰水解酶活性。因此,Gln281-> Arg取代似乎是造成血浆PAF乙酰水解酶活性降低的原因。
  • 【艾柯替尼在EGFR罕见突变的亚洲晚期非小细胞肺癌中的真实疗效和耐药机制:一项多中心研究。】 复制标题 收藏 收藏
    DOI:10.1002/cam4.2652 复制DOI
    作者列表:Lei L,Wang WX,Zhu YC,Li JL,Fang Y,Wang H,Zhuang W,Zhang YB,Wang LP,Fang MY,Xu CW,Wang XJ,Lv TF,Song Y
    BACKGROUND & AIMS: :The response to icotinib in advanced non-small cell lung cancers (NSCLC) with EGFR uncommon mutation (EGFRum) is unclear. Here we reported the efficacy and potential resistance mechanism of icotinib in Chinese EGFRum NSCLC patients. Between July 2013 and November 2016, 3117 NSCLC patients were screened for EGFRum in a multi-center study in China. Circulating tumor DNA (ctDNA) was detected and analyzed using next-generation sequencing (NGS) after progression from icotinib. The efficacy, safety and the potential resistance mechanism of icotinib were explored. After a median follow-up of 6.2 months, 69 patients (70.41%) developed disease progression, the objective rate (ORR) and disease control rate (DCR) were 13.27% and 29.59% respectively, and the median progression-free survival (PFS) was 5.5 months (95% CI: 1.2-13.0 months). Both complex-pattern with EGFR classical mutations (EGFRcm) and single-pattern have better PFS than complex-pattern without EGFRcm (median PFS was 7.2 (95% CI: 4.65-9.75), 5.2 (95% CI: 3.24-7.16) and 3.2 (95% CI: 2.97-3.44) months, respectively, P < .05); patients harboring S768I mutation had the worst PFS than others (2.0 months, P < .05). Diarrhea was the most frequent side effect (42.9%). Forty-eight (69.6%) patients developed drug resistance after 3.0 months and 81.2% of them acquired T790M mutation. Better response was observed in complex-pattern with the EGFRcm group. S768I mutation carriers may not benefit from icotinib. Acquired T790M mutation was common in icotinib-resistant EGFRum NSCLC patients.
    背景与目标: :尚不清楚EGFR罕见突变(EGFRum)的晚期非小细胞肺癌(NSCLC)对icotinib的反应。在这里,我们报道了icotinib在中国EGFRum NSCLC患者中的疗效和潜在的耐药机制。 2013年7月至2016年11月,在中国的一项多中心研究中,对3117例NSCLC患者进行了EGFRum筛查。从icotinib病情进展后,使用下一代测序(NGS)检测并分析了循环肿瘤DNA(ctDNA)。探索了icotinib的疗效,安全性和潜在的耐药机制。中位随访6.2个月后,有69名患者(70.41%)出现疾病进展,客观发生率(ORR)和疾病控制率(DCR)分别为13.27%和29.59%,中位无进展生存期(PFS) )为5.5个月(95%CI:1.2-13.0个月)。具有EGFR经典突变(EGFRcm)的复杂模式和单一模式均比没有EGFRcm的复杂模式(中位PFS为7.2(95%CI:4.65-9.75),5.2(95%CI:3.24-7.16)和3.2(95%CI:2.97-3.44)个月,P <.05);携带S768I突变的患者的PFS比其他患者差(2.0个月,P <.05)。腹泻是最常见的副作用(42.9%)。 48例(69.6%)患者在3.0个月后出现了耐药性,其中81.2%的患者获得了T790M突变。 EGFRcm组在复杂模式中观察到更好的反应。 S768I突变携带者可能无法从icotinib中获益。获得性T790M突变在依科替尼耐药的EGFRum NSCLC患者中很常见。

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