eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells

ObjectiveWe investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K)/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs).MethodsCortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.ResultsMdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.ConclusionseEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.     

Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide-binding domain 1

Misfolding and degradation of CFTR is the cause of disease in patients with the most prevalent CFTR mutation, an in-frame deletion of phenylalanine (F508del), located in the first nucleotide-binding domain of human CFTR (hNBD1). Studies of (F508del)CFTR cellular folding suggest that both intra- and inter-domain folding is impaired. (F508del)CFTR is a temperature-sensitive mutant, that is, lowering growth temperature, improves both export, and plasma membrane residence times. Yet, paradoxically, F508del does not alter the fold of isolated hNBD1 nor did it seem to perturb its unfolding transition in previous isothermal chemical denaturation studies. We therefore studied the in vitro thermal unfolding of matched hNBD1 constructs ±F508del to shed light on the defective folding mechanism and the basis for the thermal instability of (F508del)CFTR. Using primarily differential scanning calorimetry (DSC) and circular dichroism, we show for all hNBD1 pairs studied, that F508del lowers the unfolding transition temperature (T_m) by 6–7°C and that unfolding occurs via a kinetically-controlled, irreversible transition in isolated monomers. A thermal unfolding mechanism is derived from nonlinear least squares fitting of comprehensive DSC data sets. All data are consistent with a simple three-state thermal unfolding mechanism for hNBD1 ± F508del: N(±MgATP) ⇄ I_T(±MgATP) → A_T → (A_T)_n. The equilibrium unfolding to intermediate, I_T, is followed by the rate-determining, irreversible formation of a partially folded, aggregation-prone, monomeric state, A_T, for which aggregation to (A_T)_n and further unfolding occur with no detectable heat change. Fitted parameters indicate that F508del thermodynamically destabilizes the native state, N, and accelerates the formation of A_T.     

Roles of phospholipase D in phorbol myristate acetate‐stimulated neutrophil respiratory burst

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10‐fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA‐stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA‐stimulated respiratory burst. Using 1‐butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA‐stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA‐stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA‐stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA‐stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2‐dioctanoyl‐sn‐glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA‐stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA‐stimulated respiratory burst.     

尼泊尔生态系统质量评估与时空格局变化——基于参照条件的评估方法

良好的生态系统质量是维持人类社会供给需求和可持续发展目标实现的重要保障。针对尼泊尔自然地理环境复杂多样，区域间气候差异明显的特点，结合基于参照条件的评估方法可以得到生态系统质量的相对水平值，其结果能够反映出不同的变化信息。植被是区域生态系统质量变化的重要指示器，利用尼泊尔五大地理区以及四种主要植被生态系统类型划分出20个生态评估区，从表征植被生态系统的水平结构、生产功能和垂直结构3个方面计算生态参数相对密度指标（RVI），结合主成分分析法构建植被生态系统质量指数（VEQI），并以其国家自然保护区为参照，构建基于参照条件的生态系统质量评估模型，计算了尼泊尔2016和2020年基于参照条件的植被生态系统质量指数（VEQI''）并分析其生态系统质量的时空格局变化。结果表明：（1）2016至2020年，尼泊尔生态系统质量现实值VEQI的平均值增加了3.49%，总体上，在参照生态系统质量（VEQI_ref）提高（约1.41%）的背景下，生态系统质量相对水平值VEQI''增加了1.42%；（2）对于尼泊尔地区，评估区89%分位数的VEQI与其对应的国家自然保护区的参照值具有很强的相关性，总体差异较小，可以代替作为参照值；（3）从空间格局变化趋势来看，尼泊尔生态系统质量变好、基本稳定和变差的面积分别占植被生态系统总面积的74.16%、14.25%和11.59%。与数量不足、较难收集利用的野外观测台站数据相比，国家自然保护区更接近理想参照生态系统的假设，通过有限的自然保护区确定生态评估区的参照值，实现生态系统质量的快速评估，其结果具有更好的时空可比性，可以为区域生态质量变化评估及量化分析等方面提供参考。     

OpenAWSEM with Open3SPN2: A fast,flexible, and accessible framework for large-scale coarse-grained biomolecular simulations

We present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMM’s Custom Forces software framework. By utilizing GPUs, we achieve around a 30-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a single CPU core. We showcase the benefits of OpenMM’s Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsen’s early Nobel prize winning experiments by using OpenMM’s Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology.     

严重急性呼吸综合征冠状病毒2膜蛋白对宿主细胞pre-mRNA 3"UTR加工的影响

目的 研究严重急性呼吸综合征冠状病毒2（SARS-CoV-2）膜蛋白对宿主细胞mRNA前体（pre-mRNA）3"非翻译区（UTR）加工的影响。方法 本研究以人肺上皮细胞系A549为模型，利用瞬时转染在细胞内过表达SARS-CoV-2膜蛋白；利用RNA-Seq测序技术及生物信息学分析方法，系统性描绘宿主细胞选择性多聚腺苷酸化（alternative polyadenylation，APA）事件；Metascape数据库对发生显著APA变化的基因进行功能富集分析；RT-qPCR验证靶基因3"UTR长度变化；蛋白质免疫印迹（Western blot）检测目的蛋白表达水平。结果 SARS-CoV-2膜蛋白外源表达后宿主细胞内共813个基因发生显著APA变化。GO和KEGG分析显示，差异APA基因广泛参与有丝分裂细胞周期、调节细胞应激等生物过程，涉及病毒感染和蛋白质加工等。从中进一步筛选出AKT1基因，在IGV软件中显示3"UTR延长；RT-qPCR验证AKT1基因的3"UTR长度变化趋势；Western blot结果显示AKT1蛋白磷酸化水平增加。结论 SARS-CoV-2膜蛋白潜在影响宿主pre-mRNA的3"UTR加工，其中参与多种病毒性生物过程的AKT1基因 3"UTR延长，且其编码的蛋白质功能在细胞内被激活。     

CDP-Diacylglycerol Synthetase Coordinates Cell Growth and Fat Storage through Phosphatidylinositol Metabolism and the Insulin Pathway

During development, animals usually undergo a rapid growth phase followed by a homeostatic stage when growth has ceased. The increase in cell size and number during the growth phase requires a large amount of lipids; while in the static state, excess lipids are usually stored in adipose tissues in preparation for nutrient-limited conditions. How cells coordinate growth and fat storage is not fully understood. Through a genetic screen we identified Drosophila melanogaster CDP-diacylglycerol synthetase (CDS/CdsA), which diverts phosphatidic acid from triacylglycerol synthesis to phosphatidylinositol (PI) synthesis and coordinates cell growth and fat storage. Loss of CdsA function causes significant accumulation of neutral lipids in many tissues along with reduced cell/organ size. These phenotypes can be traced back to reduced PI levels and, subsequently, low insulin pathway activity. Overexpressing CdsA rescues the fat storage and cell growth phenotypes of insulin pathway mutants, suggesting that CdsA coordinates cell/tissue growth and lipid storage through the insulin pathway. We also revealed that a DAG-to-PE route mediated by the choline/ethanolamine phosphotransferase Bbc may contribute to the growth of fat cells in CdsA RNAi.     

(2-Hydroxypropyl)-β-Cyclodextrin Is a New Angiogenic Molecule for Therapeutic Angiogenesis

BackgroundPeripheral artery disease (PAD), which is caused by atherosclerosis, results in progressive narrowing and occlusion of the peripheral arteries and inhibits blood flow to the lower extremities. Therapeutic angiogenesis is a promising strategy for treating ischemia caused by PAD. Nitric oxide (NO) has been shown to be a key mediator of angiogenesis. It has been demonstrated that β-cyclodextrincan stimulate vessel growth in rabbit corneas. In this study, we assessed the mechanism of action and therapeutic potential of a new angiogenic molecule, (2-hydroxypropyl)-β-cyclodextrin (2HP-β-CD).ConclusionsTherapeutic angiogenesis by 2HP-β-CD may be beneficial to patients with PAD.