Evolutionarily Conserved Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A/B Proteins Functionally Interact with Human and Drosophila TAR DNA-binding Protein 43 (TDP-43)

Human TDP-43 represents the main component of neuronal inclusions found in patients with neurodegenerative diseases, especially frontotemporal lobar degeneration and amyotrophic lateral sclerosis. In vitro and in vivo studies have shown that the TAR DNA-binding protein 43 (TDP-43) Drosophila ortholog (TBPH) can biochemically and functionally overlap the properties of the human factor. The recent direct implication of the human heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1, known TDP-43 partners, in the pathogenesis of multisystem proteinopathy and amyotrophic lateral sclerosis supports the hypothesis that the physical and functional interplay between TDP-43 and hnRNP A/B orthologs might play a crucial role in the pathogenesis of neurodegenerative diseases. To test this hypothesis and further validate the fly system as a useful model to study this type of diseases, we have now characterized human TDP-43 and Drosophila TBPH similarity in terms of protein-protein interaction pathways. In this work we show that TDP-43 and TBPH share the ability to associate in vitro with Hrp38/Hrb98DE/CG9983, the fruit fly ortholog of the human hnRNP A1/A2 factors. Interestingly, the protein regions of TDP-43 and Hrp38 responsible for reciprocal interactions are conserved through evolution. Functionally, experiments in HeLa cells demonstrate that TDP-43 is necessary for the inhibitory activity of Hrp38 on splicing. Finally, Drosophila in vivo studies show that Hrp38 deficiency produces locomotive defects and life span shortening in TDP-43 with and without animals. These results suggest that hnRNP protein levels can play a modulatory role on TDP-43 functions.     

低温对龙井43种胚基因表达效应的初步研究

为了探讨低温对茶树种子特定基因表达的效应,以便为茶树种子长期低温储存提供分子生物学方面的参考依据,采用cDNA-AFLP方法对茶树无性系龙井43种子经超低温（-70 ℃）储存1年后种胚的基因转录活性进行了初步分析,结果显示：64对引物组合共产生了132条PCR产物.对其中的15条进行克隆测序,最终获得8条差异片段.在GenBank进行序列分析,发现有6条与已知功能基因相关,其中包括参与植物转运、转录、能量产生及老化相关的蛋白,2条在GenBank数据库中未找到相似序列.     

Cation permeation through connexin 43 hemichannels is cooperative, competitive and saturable with parameters depending on the permeant species

Kinetics of permeation through connexin 43-EGFP hemichannels (Cx43-EGFP HCs) were evaluated in divalent cation-free solutions, which enhance HC open probability and thus, allow measurements during initial velocity. Three cations that become fluorescent upon binding to intracellular nucleic acids [ethidium (Etd), propidium (Prd) and 4′,6-diamidino-2-phenylindole (DAPI)] and Cx43-EGFP or Cx43 wild type HeLa cell transfectants (Cx43-EGFP- and Cx43-WT-HeLa cells, respectively) were used. Levels of Cx43-EGFP at the cell periphery and rate of dye uptake were directly related. The rate of uptake of each dye reached saturation consistent with a facilitated transport mechanism. Before saturation, the relation between rate of uptake and concentration of each dye was sigmoidal with Hill coefficients >1, indicating positive cooperativity of transport at low concentrations. The maximal rate of Etd uptake was not affected by the presence of DAPI and vice versa, but under each condition the apparent affinity constant of the main permeant molecule increased significantly consistent with competitive inhibition or competition for binding sites within the channel. Moreover, Cx43-EGFP and Cx43-WT HCs had similar permeability properties, indicating that EGFP bound to the C-terminal of Cx43 does not significantly alter the permeability of Cx43 HCs to positively charged molecules. Thus, competitive inhibition of permeation through hemichannels might contribute to cellular retention of essential molecules and/or uptake inhibition of toxic compounds.     

O‐GlcNAcylation of TDP‐43 suppresses proteinopathies and promotes TDP‐43’s mRNA splicing activity

Pathological TDP‐43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD‐TDP); however, how TDP‐43 aggregation and function are regulated remain poorly understood. Here, we show that O‐GlcNAc transferase OGT‐mediated O‐GlcNAcylation of TDP‐43 suppresses ALS‐associated proteinopathies and promotes TDP‐43''s splicing function. Biochemical and cell‐based assays indicate that OGT''s catalytic activity suppresses TDP‐43 aggregation and hyperphosphorylation, whereas abolishment of TDP‐43 O‐GlcNAcylation impairs its RNA splicing activity. We further show that TDP‐43 mutations in the O‐GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP‐43 overexpression in Drosophila motor neurons. We finally demonstrate that O‐GlcNAcylation of TDP‐43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. Our findings suggest that O‐GlcNAcylation might be a target for the treatment of TDP‐43‐linked pathogenesis.     

Kinetics of Protein-Protein Interactions of Connexins: Use of Enzyme Linked Sorbent Assays

Determination of the protein-protein interactions of connexins has become a rapidly expanding field of research. While there are multiple methods of determining the identity of binding partners, determination of the strengths of interactions is not as simple. Here we describe the use of the in vitro method Enzyme Linked Sorbent Assay (ELSA) to compare binding affinities of known protein partners for Connexin43. We used the binding of Cx43 Carboxyl Terminal domain to the PDZ-2 domain of Zonula Occludens-1 and to the SH3 domain of c-Src. In the ELSA assay we found that while the binding of the SH3 domain of c-Src is pH-dependent, the interaction of the PDZ domain of ZO-1 is not. These data confirm findings using Surface Plasmon Resonance (1) and indicate that ELSA can be a useful tool in determining the kinetics of protein-protein interactions.     

三氧化二砷对鼻咽癌细胞Cx43表达的影响

目的:探讨三氧化二砷(As2O3)抑制鼻咽癌的作用机制。方法:采用流式细胞仪(FCM)、激光扫描共聚焦显微镜(LSCM)和荧光技术,检测人鼻咽癌细胞株(CNE1)经As2O3诱导后细胞连接蛋白43(Cx43)表达的变化。结果:FCM显示,经过浓度为4μmol L的As2O3处理后,其Cx43阳性细胞计数率明显升高,与未经As2O3处理和经2μmol L的As2O3处理的CNE1比较,其差异具有非常显著性意义(p<0.01);LSCM图像观察到,经4μmol LAs2O3处理后,标记Cx43的绿色荧光显著增强,集中分布于细胞膜。结论:较高剂量浓度的As2O3能提高鼻咽癌细胞Cx43的表达率,升高细胞膜Cx43的含量。As2O3抑制鼻咽癌细胞生长的作用机制之一,是通过恢复细胞间隙连接通讯功能来实现的。     

Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts

To examine the role of gap junctions in cell senescence, the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts.Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore, cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis, p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin(10mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis, elevation of p53 expression, loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.     

Connexin31 显性听力下降相关突变体研究

Cx31 突变可以导致常染色体显性听力下降、常染色体隐性听力下降、周围神经疾病伴听力丧失,以及变性红皮肤病角化病,其导致不同疾病的机理一直是研究的重点 . 利用定点突变技术 (site-directed mutagenesis, SDM) 构建 connexin31 显性听力下降相关突变体 Cx31R180X , Cx31E183K 并插入到真核表达载体 pEGFP-N1 ,转染 HeLa 细胞,转染 Cx31R180X-pEGFP-N1 和 Cx31E183K-pEGFP-N1 Cx31 突变体质粒的 HeLa 细胞质膜上没有出现斑块状染色和聚集现象,分别用内质网染料 conA 和高尔基体染料 WGA 进行免疫荧光染色,结果显示 Cx31 显性听力下降相关突变体蛋白主要分布在细胞质内,且大部分定位在内质网和高尔基体上 . 同时分别用抗 Cx31 和抗 GFP 抗体进行蛋白质印迹检测,证实 Cx31R180X , Cx31E183K 在转染的 HeLa 细胞中都有表达 . 研究发现 Connexin31 显性耳聋相关突变体 Cx31R180X , Cx31E183K 不能正常地形成间隙连接通道,这与 connexin31 EKV ( 变性红皮肤病角化病 ) 相关突变体能够运输到细胞膜上形成间隙连接通道的报道不相同,提示 connexin31 不同部位突变导致不同疾病的致病机理可能不一样 , 从而为解释“ one connexin two diseases ”提供分子水平的依据 .     

Influence of tris(2,3-dibromopropyl) isocyanurate on the expression of photosynthesis genes of Nannochloropsis sp.

The proteins encoded by psaA and psaB form a heterodimer, an essential compound of photosystem; while the protein encoded by psbC binds with chlorophyll a in photosystem II, serving as antennae in photosystem. Here we report that a heterocyclic brominated flame retardant, tris(2,3-dibromopropyl) isocyanurate (TBC), inhibited the expression of psaA and psbC, then leads to the decrease of Nannochloropsis sp.'s growth biomass. TBC exposure inhibited the expression of psaA and psbC at 10, 100 ng/mL slightly and 1000 ng/mL significantly. In addition, TBC was found to slow down the growth of Nannochloropsis sp. at concentrations ranging from 10 to 1000 ng/mL. These results indicated that TBC influenced both photosynthesis and growth performance of Nannochloropsis sp.     

Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (at 15 min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.