Development of dual casein kinase 1δ/1ε (CK1δ/ε) inhibitors for treatment of breast cancer

Casein kinase 1δ/ε have been identified as promising therapeutic target for oncology application, including breast and brain cancer. Here, we described our continued efforts in optimization of a lead series of purine scaffold inhibitors that led to identification of two new CK1δ/ε inhibitors 17 and 28 displaying low nanomolar values in antiproliferative assays against the human MDA-MB-231 triple negative breast cancer cell line and have physical, in vitro and in vivo pharmacokinetic properties suitable for use in proof of principle animal xenograft studies against human cancers.     

α1A-Adrenergic receptor prevents cardiac ischemic damage through PKCδ/GLUT1/4-mediated glucose uptake

While α₁-adrenergic receptors (ARs) have been previously shown to limit ischemic cardiac damage, the mechanisms remain unclear. Most previous studies utilized low oxygen conditions in addition to ischemic buffers with glucose deficiencies, but we discovered profound differences if the two conditions are separated. We assessed both mouse neonatal and adult myocytes and HL-1 cells in a series of assays assessing ischemic damage under hypoxic or low glucose conditions. We found that α₁-AR stimulation protected against increased lactate dehydrogenase release or Annexin V⁺ apoptosis under conditions that were due to low glucose concentration not to hypoxia. The α₁-AR antagonist prazosin or nonselective protein kinase C (PKC) inhibitors blocked the protective effect. α₁-AR stimulation increased ³H-deoxyglucose uptake that was blocked with either an inhibitor to glucose transporter 1 or 4 (GLUT1 or GLUT4) or small interfering RNA (siRNA) against PKCδ. GLUT1/4 inhibition also blocked α₁-AR-mediated protection from apoptosis. The PKC inhibitor rottlerin or siRNA against PKCδ blocked α₁-AR stimulated GLUT1 or GLUT4 plasma membrane translocation. α₁-AR stimulation increased plasma membrane concentration of either GLUT1 or GLUT4 in a time-dependent fashion. Transgenic mice overexpressing the α_1A-AR but not α_1B-AR mice displayed increased glucose uptake and increased GLUT1 and GLUT4 plasma membrane translocation in the adult heart while α_1A-AR but not α_1B-AR knockout mice displayed lowered glucose uptake and GLUT translocation. Our results suggest that α₁-AR activation is anti-apoptotic and protective during cardiac ischemia due to glucose deprivation and not hypoxia by enhancing glucose uptake into the heart via PKCδ-mediated GLUT translocation that may be specific to the α_1A-AR subtype.     

Casein Kinase 1δ-dependent Wee1 Protein Degradation

Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation. Small-molecule CK1δ inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1δ in controlling the cell cycle.     

δN and δC analysis of a Posidonia sinuosa seagrass bed

Potential food sources and dominant invertebrates and fishes were collected for the examination of variability in ¹³C/¹²C and ¹⁵N/¹⁴N to determine the sources of carbon available to consumers within a Western Australian Posidonia sinuosa-dominated seagrass bed. Autotrophs showed a wide distribution of δ¹³C values, with P. sinuosa at −11.3 ± 0.8‰ and macroalgae ranging from −16.6 to −31.7‰. This variation allowed us to successfully identify macroalgae as the main contributor of carbon to the trophic structure, although no distinction could be made between epiphytic macroalgae on seagrass, or allochthonous macroalgal sources. The range in δ¹⁵N ratios among potential food items at the trophic base was too small to make it useful as tracer of nitrogen flow pathways, but it consistently increased from macrophytes and detritus (4.1–6.8‰), to invertebrates (5.7–7.4‰) located near the middle of the food web, to fishes (8.3–11.9‰), with piscivorous species such as Leviprora inops generally having a higher ¹⁵N. δ¹³C of seston (−12.8‰) and sedimentary organic matter (−8.7‰) indicate that seagrass material is the main contributor to these two carbon pools, and that very little of it contributes to animal biomass.     

疏水色谱法制备缺失δ亚基的CF_1(-δ)

PreperationofCF_1DeficientintheδSubunitbyHydrophobicColumnChro－matographyRENHut－Miao，WEIJia－Mian（ShanghaiInstituteofPlantPhysiology,TheChineseAcademyofSciences.Shanghai200032）叶绿体类囊体膜上H－ATPase在光合作用的能量转换过程中起着重要的作用。光合磷酸化就是H”－ATPase利用光合电子传递产生的跨膜电化学质子梯度，把ADP和Pi合成ATP的过程。但是人们对H”－ATPase催化合成ATP的机理、CF；和CF。的连接、各个亚基的功能以及该酶的调节等几个关键性的问题还没有了解得十分清楚。而制备获得缺…     

TNF-α Mediates PKCδ/JNK1/2/c-Jun-Dependent Monocyte Adhesion via ICAM-1 Induction in Human Retinal Pigment Epithelial Cells

Retinal inflammatory diseases induced by cytokines, such as tumor necrosis factor-α (TNF-α) are associated with an up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the retinal pigment epithelial cells (RPECs). Retinal pigment epithelium (RPE) is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). However, the detailed mechanisms of TNF-α-induced ICAM-1 expression are largely unclear in human RPECs. We demonstrated that in RPECs, TNF-α could induce ICAM-1 protein and mRNA expression and promoter activity, and monocyte adhesion. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of PKCs (Ro318220), PKCδ (Rottlerin), MEK1/2 (U0126), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of TNFR1, TRAF2, JNK2, p42, or c-Jun. We showed that TNF-α could stimulate the TNFR1 and TRAF2 complex formation. TNF-α-stimulated JNK1/2 was also reduced by Rottlerin or SP600125. However, Rottlerin had no effect on TNF-α-induced p42/p44 MAPK phosphorylation. We observed that TNF-α induced c-Jun phosphorylation which was inhibited by Rottlerin or SP600125. On the other hand, TNF-α-stimulated ICAM-1 promoter activity was prominently lost in RPECs transfected with the point-mutated AP-1 ICAM-1 promoter plasmid. These results suggest that TNF-α-induced ICAM-1 expression and monocyte adhesion is mediated through a TNFR1/TRAF2/PKCδ/JNK1/2/c-Jun pathway in RPECs. These findings concerning TNF-α-induced ICAM-1 expression in RPECs imply that TNF-α might play an important role in ocular inflammation and diseases.     

Mg2+依赖性蛋白磷酸酶1δ的生物信息学分析

Mg2+依赖性蛋白磷酸酶1δ(protein phosphatase magnesium-dependent 1δ,PPM1D)作为肝癌潜在的预后标志物和治疗靶点,其致癌机制和预后价值仍未完全阐明。为了全面认识PPM1D,使用生物信息学方法,对PPM1D蛋白的序列同源性、组织表达、亚细胞定位、理化性质、空间结构及蛋白质相互作用网络进行分析。结果表明:人PPM1D基因编码605个氨基酸组成的多肽,与物种进化程度一致,属于PP2C蛋白超家族,是碱性不稳定的亲水蛋白,无信号肽和跨膜区域;PPM1D蛋白主要定位于细胞核内,其主要二级结构为随机卷曲,存在磷酸化、乙酰化、甲基化和泛素化位点,与PPM1D相互作用的蛋白主要是细胞周期检查点蛋白和细胞损伤修复相关蛋白。根据分析结果阐述了PPM1D蛋白与癌症的相关性以及PPM1D蛋白作为癌症标志物的理论基础,为进一步研究该蛋白及其参与的信号通路提供一定的借鉴和参考。     

Variation among diets in discrimination of δC and δN in the amphipod Allorchestes compressa

Discrimination of stable isotopes of carbon (δ¹³C) and nitrogen (δ¹⁵N) was examined for the amphipod Allorchestes compressa Dana using controlled laboratory experiments. Amphipods were fed exclusively on single diets (fresh or decomposed macroalgae or seagrass) for three weeks. Macrophyte type (i.e. seagrass, brown algae or red algae) had a greater influence on the stable isotope ratios of A. compressa than the state of decomposition of the macrophyte material. The experiments revealed that δ¹³C in A. compressa stabilised at values lower than those of the diets, which contrasts to the general assumption that consumer-diet discrimination of δ¹³C ranges from 0 to + 1‰. Amphipods fed on seagrass yielded the lowest δ¹³C values, which were 9 to 10‰ lower than their diet, while amphipods fed on macroalgae had values 2 to 4‰ lower than their diet. In addition, contrary to the general assumption that consumer-diet discrimination of δ¹⁵N ranges from + 3 to + 5‰, discrimination of δ¹⁵N was as low as − 1 and + 1 when A. compressa was fed on brown and red algae, respectively, but as high as + 3‰ when fed on seagrass. The results show that discrimination of stable isotopes of carbon and nitrogen can vary considerably depending on the food source, demonstrating that validation of assumptions about discrimination are critical for interpreting stable isotope data from field studies.     

抗α2δ1抗体/NanoLuc融合蛋白的制备及其特性初步验证

肿瘤干细胞的存在被认为是肿瘤耐药和复发转移的根本原因,因此研发肿瘤干细胞的活体标记示踪技术动态监测该类细胞的存在具有重要的科学价值。实验室前期发现电压门控钙离子通道α2δ1亚基是肝细胞癌干细胞的特异表面标志物,文中研究探讨利用针对α2δ1的单克隆抗体1B50-1的单链可变域与新型荧光素酶NanoLuc形成融合蛋白1B50-1scFv-NanoLuc,在体外验证其对α2δ1阳性肝癌细胞结合的特性和荧光素酶活性。1B50-1scFv和NanoLuc序列通过重叠PCR技术扩增获得1B50-1scFv-NanoLuc的融合片段,并在C端添加Flag标签肽 (命名为1B50-1scFv-NanoLucFlag),然后采用常规DNA克隆技术将融合片段克隆到真核表达载体。利用聚乙烯亚胺(Polyethyleneimine,PEI) 将1B50-1scFv-NanoLucFlag真核表达载体转染至悬浮型人胚肾293细胞 (FreeStyle 293F)。蛋白免疫印记实验证明融合蛋白在FreeStyle 293F细胞培养上清中表达,其分子量约为50 kDa。融合蛋白经ANTI-FLAG? M2亲和层析柱纯化后,经流式细胞仪法测定融合蛋白1B50-1scFv-NanoLucFlag对高表达α2δ1的Hep-12肝癌细胞具有高亲和活性。进一步,1B50-1scFv-NanoLucFlag与高表达α2δ1的肝癌细胞结合后显示出强的荧光素酶活性。这些结果表明融合蛋白1B50-1scFv-NanoLucFlag可用于α2δ1阳性肝癌干细胞的标记并可通过荧光素酶化学发光法进行示踪。     

基于δD和δ18O的青海湖流域芨芨草水分利用来源变化研究

水分条件是限制干旱半干旱地区植物生长重要的生态因子,为了揭示青海湖流域典型生态系统下芨芨草植物的水分利用来源及其如何响应水分条件的变化,选择了自然和干旱控制条件下芨芨草植物,通过测定芨芨草植物茎水和各潜在水源(土壤水、地下水及降水)中δD、δ~(18)O组成,并利用直接比较分析法和多源混合模型计算芨芨草植物对土壤水的利用比例。研究结果表明:表层土壤水分和土壤水中δD、δ~(18)O值表征出较大波动范围,其直接受降水和蒸发作用影响,土壤蒸发线的斜率和截距明显小于大气水线斜率和截距,表明土壤水中同位素组成经历了强烈的蒸发分馏过程,而芨芨草茎水中δD、δ~(18)O值都集中分布土壤水蒸发线附近,说明芨芨草根系主要利用不同深度的土壤水。自然条件下芨芨草在生长季初期(6月)利用表层土壤水(0—10cm,45.1%),8—9月份大降水事件影响土壤含水量和同位素组成,降水入渗深度较深且芨芨草根系对土壤水分吸收的比例相差不大,表明根系在土壤含水量较高时均能吸水不同深度土壤水。在干旱控制条件下芨芨草在7月初主要利用表层土壤水(0-30cm),随着表层土壤水分的减少,根系吸收深度转向较深土壤层,而灌溉后表层土壤水分明显增加,其吸收深度又转向表层,表明芨芨草根系吸收深度能敏感地响应土壤水分的变化。另外还发现芨芨草在生长季内并未直接利用地下水。     

内蒙古草原常见植物叶片δ13C和δ15N对环境因子的响应

在中国东北样带沿线的内蒙古草原地区采集了一些常见植物的叶片样品,并测定其δ¹³C和δ¹⁵N值,分析了其统计学特征以及对环境因子(年平均降雨量和温度)的响应模式。发现东北样带草原区同时存在C₃和C₄两种不同光合途径的植物,但是C₃植物占主导地位,C₄植物数量有限。C₃植物叶片δ¹³C随着年平均降雨量和年平均温度的升高而显著降低,反映了此区域C₃植物δ¹³C受控于降水量和温度。C₄植物的叶片δ¹³C值随着降雨量的增多而有轻微升高的趋势,但是C₄植物的叶片δ¹³C值对年平均温度的响应不敏感。不论对C₃植物还是C₄植物而言,叶片δ¹⁵N都随降雨量增加而显著降低,即干旱区的植物叶片δ¹⁵N大于湿润地区,这说明降水是影响植物叶片δ¹⁵N的一个重要因素。然而两者叶片δ¹⁵N对温度的响应不敏感。     

栓皮栎叶片δ13C和δ15N的纬向趋势及其影响因子

以东亚广布种栓皮栎为例,通过对南北样带(26°-40° N)上7个群体的调查取样,并结合2个生活史阶段,探讨栓皮栎碳氮同位素比值随纬度环境的变异规律.结果表明: 在纬向梯度上,栓皮栎叶片δ¹³C和δ¹⁵N随纬度的增加分别呈现非线性的增加和下降趋势,且成年树叶片δ¹³C和δ¹⁵N均显著高于幼树;同时,树龄和纬度对叶片δ¹⁵N和δ¹³C均无显著交互作用,表明栓皮栎幼树和成年树纬向环境变化的响应较为一致.随机森林模型结果显示,栓皮栎叶片δ¹⁵N主要受土壤养分,如土壤有机质、磷和氮含量的影响,而叶片δ¹³C主要受水分因子,如空气相对湿度、降水量等的影响.     

CMV-Independent Lysis of Glioblastoma by Ex Vivo Expanded/Activated Vδ1+ γδ T Cells

Vδ2^neg γδ T cells, of which Vδ1+ γδ T cells are by far the largest subset, are important effectors against CMV infection. Malignant gliomas often contain CMV genetic material and proteins, and evidence exists that CMV infection may be associated with initiation and/or progression of glioblastoma multiforme (GBM). We sought to determine if Vδ1+ γδ T cells were cytotoxic to GBM and the extent to which their cytotoxicity was CMV dependent. We examined the cytotoxic effect of ex vivo expanded/activated Vδ1+ γδ T cells from healthy CMV seropositive and CMV seronegative donors on unmanipulated and CMV-infected established GBM cell lines and cell lines developed from short- term culture of primary tumors. Expanded/activated Vδ1+ T cells killed CMV-negative U251, U87, and U373 GBM cell lines and two primary tumor explants regardless of the serologic status of the donor. Experimental CMV infection did not increase Vδ1+ T cell - mediated cytotoxicity and in some cases the cell lines were more resistant to lysis when infected with CMV. Flow cytometry analysis of CMV-infected cell lines revealed down-regulation of the NKG2D ligands ULBP-2, and ULBP-3 as well as MICA/B in CMV-infected cells. These studies show that ex vivo expanded/activated Vδ1+ γδ T cells readily recognize and kill established GBM cell lines and primary tumor-derived GBM cells regardless of whether CMV infection is present, however, CMV may enhance the resistance GBM cell lines to innate recognition possibly contributing to the poor immunogenicity of GBM.     

δ-Aminolaevulinate biosynthesis in the cyanobacterium Synechococcus 6301

The cyanobacterium (blue-green alga) Synechococcus 6301 incorporated a large amount of isotope from [1-¹⁴C] and [2-¹⁴C]acetate into phaeophorbide a obtained from chlorophyll a and into glutamatein cell protein; very little radioactivity was present in aspartate in cell protein. This distribution of isotope indicates that aspartate and the tetrapyrrole of chlorophyll a are not derived from a common C₄, precursor. The ratios of the specific radioactivities of phaeophorbide a to glutamate for organisms grown in the presence of 1-¹⁴C] and [2-⁴C ] acetate were 2.5:1 and 10:1 respectively. These are close to the theoretical values for the C₅, route to δ-aminolaevulinate which indicates that this is the only pathway to the tetrapyrrole precursor in Synechococcus 6301.     

Termination of early pregnancy by ONO-802 (16,16-Dimethyl-trans-δ2-PGE1 methyl ester)

ONO-802 was infused into th euterine cavity for the termination of early pregnancy in 45 healthy volunteers. Forty two (93%) of the 45 cases had compelete abortions and two had incomplete abortions.Nine (20%) of the 45 volunteers complained of nausea, 7 (16%) vomited and 5 (11%) complained of abdominal pain. Neither diarrhea nor weakness was observed.These results suggest that ONO-802 is more acceptable for the termination of early pregnancy than is PGF_2α.