Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

Fluorescence litime imaging(FLIM)is an effective noninvasive bioanalytical tol based onmeasuring fuorescent lifetime of fuorophores.A growing number of FLIM studies utilizes ge-netically engineered fluorescent proteins targeted to specific subcellular structures to probe localmolecular environment,which opens new directions in cell science.This paper highlights theunconventional applications of FLIM for studies of molecular processes in diverse organelles oflive cultured cells.

Simultaneous Cell Disruption and Aqueous Two-Phase Extraction for Isolation of Intracellular Recombinant Proteins

A new technique was developed for the integrated processing of cell disruption and aqueous two-phase extraction in a high-speed bead mill to separate intracellular proteins form microbial cells. The process was narned as simultanecus cell disruption and aqueous two-phase extraction (ADATE). Advantages, such as high cell disruption efficiency, biochemical activities proservation of proteins, cell debris elimination, and preliminary puriffcation of the target protein were being clairmed. When this technique was employed for isolating recombinant Tumor Necrois Factor (TNF) from E.coli, overall protein codcentration and TNF activity were found to have been increased. More than 85% of TNF was partitioned into the top phase and all cell debris were in the bottom phase. The partition coefficinet was greater than 3 and the TNF puriflcation fsctop was greater than 6. It is zhown that less separation steps were being utilized in the new techniqne, meaning a reduction in separation time and less process extractors required.

Micro and Nanotechnology for Intracellular Delivery Therapy Protein

Proteins therapy is of great importance in the treatment of protein deficiency disease. Most human diseases are related to the malfunctioning of one or more proteins. The most effective and direct approach is protein therapy, which delivers the proteins into the target cell to replace the dysfunction protein and maintain the balance of organism. However, clinical application is frequently hampered by various biological barriers to their successful delivery. This review aims to discuss the recent advances about microparticles and nanoparticles fabricated using micro and nanotechnology for intracellular delivery therapy protein and give some suggestions about the promising delivery system.

Hepatitis B Virus X Protein Upregulates Intracellular Calcium Signaling by Binding C-terminal of Orai1 Protein

Hepatitis B virus X（HBx） protein plays a pivotal role in the development of hepatitis B virus（HBV）-associated hepatocellular carcinoma.Although regulation of cytosolic calcium is essential for HBV replication and is mediated by HBx protein,the mechanism of HBx protein regulating intracellular calcium level remains poorly understood.The present study examined whether HBx protein elevated the intracellular calcium through interacting with storeoperated calcium entry（SOCE） components,Orai1 and stromal interaction molecule 1,and then identified the targets of HBx protein,with an attempt to understand the mechanism of HBx protein upsetting intracellular calcium homeostasis.By employing co-immunoprecipitation and GST-pull-down assay,we found that Orai1 protein interacted with HBx protein,and the C-terminus of Orai1 was implicated in the interaction.Confocal microscopy also revealed that HBx protein could co-localize with full-length Orai1 protein in HEK293 cells.Moreover,live cell calcium imaging exhibited that HBx protein elevated intracellular calcium,possibly by binding to SOCE components.Our results suggest that HBx protein binds to STIM1-Orai1 complexes to positively regulate the activity of plasma membrane store-operated calcium channels.

Effect of Low Dose Radiation on Intracellular Calcium and Protein Kinase C in Lymphocytes

It is first reported in the present paper that whole-body irradiation (WBI) with low dose X-rays could increase intracellular calcium ions ([Ca2+]i) and stimulate protein kinase C (PKC) activity of mouse lymphocytes. Following WBI of male Kunming micc With 75 mGy X-rays at a dose rate of 12.5 mGy/min the mobilization of [Ca2+]i with Con A in CD4+ and CD8+ Cells in the thymus and spleen was potentiated and the amplitude of [Ca2+], mobilization in thymocytes in response to anti-CD3 monoclonal antibody increased with time from 4 to 24 h following low dose radiation. The PKC activity in the homogenate of spleen was markedly stimulated 12 h after WBl with 75 mGy, reaching its peak value at 24-48 h and coming down to lower than normal on day 7. However, the PKC activity in the separated T lymphocytes reached its peak value at 12 h and that in the B lymphocytes reached its peak value on day 4, both coming down to below control on day 7. The implications of this facilitation of signal transduction in T lymphocytes in the mechanism of immunoenhancement after low dose radiation were discussed

Two memory associated genes regulated by amyloid precursor protein intracellular domain Novel insights into the pathogenesis of learning and memory impairment in Alzheimer's disease

In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein intracellular domain binding sites in chromatin DNA from hippocampal neurons of rats, and to screen out five putative genes associated with the learning and memory functions. The promoter regions of the calcium/calmodulin-dependent protein kinase II alpha and glutamate receptor-2 genes were amplified by PCR from DNA products immunoprecipitated by amyloid precursor protein intracellular domain. An electrophoretic mobility shift assay and western blot analysis suggested that the promoter regions of these two genes associated with learning and memory were bound by amyloid precursor protein intracellular domain （in complex form）. Our experimental findings indicate that the amyloid precursor protein intracellular domain is involved in the transcriptional regulation of learning- and memory-associated genes in hippocampal neurons. These data may provide new insights into the molecular mechanism underlying the symptoms of progressive memory loss in Alzheimer＇s disease.

Optogenetic activation of intracellular signaling based on light-inducible protein-protein homo-interactions

Dynamic protein-protein interactions are essential for proper cell functioning.Homointeraction events—physical interactions between the same type of proteins—represent a pivotal subset of protein-protein interactions that are widely exploited in activating intracellular signaling pathways.Capacities of modulating protein-protein interactions with spatial and temporal resolution are greatly desired to decipher the dynamic nature of signal transduction mechanisms.The emerging optogenetic technology,based on genetically encoded light-sensitive proteins,provides promising opportunities to dissect the highly complex signaling networks with unmatched specificity and spatiotemporal precision.Here we review recent achievements in the development of optogenetic tools enabling light-inducible protein-protein homo-interactions and their applications in optical activation of signaling pathways.

Intracellular sorting pathways of the amyloid precursor protein provide novel neuroprotective strategies

Alzheimer＇s disease（AD）is the most common cause of senile dementia.It is characterized by the formation of plaques mainly composed of the amyloid-beta peptide（Aβ）.Diverse lines of evidence support the notion that accumulation of Aβis a primary cause of AD pathogenesis（Huang and Mucke,2012）.Amyloid precusor protein（APP）processing is dependent on its subcelluar trafficking pathway：Aβis derived from APP by proteolyric processing.

Characterization of kinesin-like proteins in silkworm posterior silkgland cells

Kinesins 是涉及各种各样的细胞内部的运输的基于微导管的马达。神经原，鞭打的房间，和颜料房间传统地被用作模型系统学习 kinesins 的细胞的功能。这里，我们报导蚕以后的 silkgland (PSG ) ，为细胞内部的运输和丝心蛋白的有效分泌物与一个广泛的 endomembrane 系统专业化房间，，为 kinesin 学习的一个新奇模型。在 PSG 房间调查驾驶 kinesin 的细胞内部的运输，我们克隆五蚕象 kinesin 一样蛋白质(KLP ) ， BmKinesin-1， BmKinesin-6， BmKinesin-7， BmKinesin-13，和 BmKinesin-14A。我们由相对即时 PCR 并且西方的弄污决定了他们的表示模式。Immunofluorescence 显微镜学与微导管验证了他们的 colocalization。由联合下拉试金， LC-MS/MS，和西方的弄污分析，我们在 PSG，包括的包含丝心蛋白的小粒和联系 exuperantia 的 ribonucleoprotein (RNP ) 建筑群识别了 BmKinesin-1 的许多潜在的货物。而且， BmKinesin-13 overexpression 在 BmN 细胞破坏了微导管网络，它与在在另外的有机体调整微导管动力学的 Kinesin-13 的一个角色一致。根据这些结果，我们断定 PSG 可能在阐明有优点在能分泌的纸巾的细胞内部的运输的机制并且能为 kinesin 研究担任一个潜在的模型。

Differential activation of mitogen-activated protein kinases by γ-irradi-ation in IEC-6 cells: Role of intracellular Ca^(2+)

Abstract Objective:To explore the effects of γ-irradiation on mitogen-activatedprotein kinases(MAPKs) and role intracellular calcium in this event in intestinal epithelial cell line 6(IEC-6 cells).Methods:After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca^2+ chelator were exposed to γ-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry.Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting.Results:In response to γ-irradiation,phosphorylation of ERK was not significantly observed ,while the levels of phos-phorylated c-Jun NH2-terminal kinase(JNK) and p38 MAPK were increased in 30 min and reached the peak 2h after exposure to 6Gy γ-irradiation,though the cell viability was significantly lowered 12h.On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK.Chelation of in-tracellular Ca^2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca^2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.

非洲猪瘟病毒CD2v蛋白胞内域与胞外域的原核表达及其抗原性分析

【目的】应用大肠杆菌表达系统表达非洲猪瘟病毒(African swine fever virus,ASFV)CD2v蛋白胞外域(N-端,CD2v-N)与胞内域(C-端,CD2v-C)并分析两者的抗原性,为ASFV检测方法的研发提供试验依据和指导。【方法】构建CD2v-N和CD2v-C的重组原核表达质粒,在体外表达并纯化CD2v-N和CD2v-C蛋白,通过Western blotting对表达蛋白进行鉴定;用表达的CD2v-N和CD2v-C蛋白分别肌内接种免疫新西兰大白兔,共免疫3次,每次间隔2周;每次免疫后14 d,应用ELISA方法测定CD2v-N或CD2v-C的特异性抗体水平;用纯化后的CD2V-N和CD2v-C蛋白作为包被抗原,通过ELISA方法检测95份ASFV感染猪的血清CD2v-N或CD2v-C特异性抗体,比较CD2v-N和CD2v-C在猪体内诱导的免疫反应水平。【结果】重组蛋白CD2v-N和CD2v-C分别以包涵体和可溶性形式表达,分子质量分别为22.9和34.0 ku,均能与猪抗ASFV血清特异性结合;用CD2v-N蛋白首免家兔后14 d,血清中未检测到特异性抗体,而在CD2v-C首免的家兔血清中检测到了高滴度的特异性抗体;三免后14 d,CD2v-N和CD2v-C蛋白免疫家兔的血清特异性抗体效价分别为1∶125000和1∶107;ELISA检测结果显示,ASFV感染猪的血清中CD2v-C特异性抗体水平显著高于CD2v-N(P<0.05)。【结论】本研究表达了ASFV CD2v蛋白胞内域和胞外域,前者的抗原性比后者高,CD2v胞内域作为ASFV免疫学检测技术研究与开发的靶标更具优势。该研究结果对ASFV检测方法的研究与开发具有重要指导意义。

金黄色葡萄球菌巨噬细胞胞内生存关键分泌蛋白筛选体系的构建

目的建立金黄色葡萄球菌(Staphylococcus aureus,S.aureus)巨噬细胞胞内感染状态下分泌蛋白高通量筛选体系,探索金葡菌胞内生存所需关键分泌蛋白。方法利用课题组建立的金葡菌分泌蛋白真核系统表达载体库,通过DNA转染表达技术,得到能够在细胞内表达金葡菌分泌蛋白的RAW264.7巨噬细胞阵列。金葡菌感染RAW264.7后,清除胞外菌,观察金葡菌胞内生存情况。最后通过构建相应分泌蛋白的过表达及敲除金葡菌验证筛选结果。结果通过探索RAW264.7质粒转染剂量、金葡菌感染复数(multiplicity of infection,MOI)以及感染时间,确立筛选体系的最优质粒转染剂量为1.0μg/孔、MOI为1.0、感染时间为4 h。筛选结果结合相应分泌蛋白过表达和敲除菌株验证,成功筛选出hypothetical protein、Serine protease E等分泌蛋白具有促进胞内金葡菌生存功能。结论成功构建金葡菌巨噬细胞胞内生存关键分泌蛋白筛选体系。

多囊卵巢综合征患者卵巢颗粒细胞胰岛素抵抗的相关信号通路

胰岛素抵抗(insulin resistance,IR)与多囊卵巢综合征(polycystic ovary syndrome,PCOS)的发生发展有关,而与IR相关的胰岛素信号通路异常会引起卵巢局部代谢紊乱,进而影响卵泡发育及卵子质量。PCOS卵巢颗粒细胞中胰岛素作用的经典通路,包括磷脂酰肌醇3激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)通路和丝裂原激活的蛋白激酶(mitogen-activated protein kinase,MAPK)/细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)通路;与炎症相关的通路包括Toll样受体(Toll-like receptor,TLR)/核因子κB(nuclear factor-κB,NF-κB)通路等;氧化应激相关通路包括核转录因子红系2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)/血红素加氧酶-1(heme oxygenase-1,HO-1)途径和GTP酶免疫相关蛋白7(GTPase of immunity-associated protein 7,GIMAP7)/音猬因子(sonic hedgehog,SHH)途径;与脂质代谢相关的通路如激活转录因子4(activating transcription factor 4,ATF4)通路等。明确PCOS患者卵巢颗粒细胞中IR相关信号转导通路,增进PCOS病理生理机制的认识,进一步为PCOS的治疗提供新思路。

直肠癌患者的血清C型凝集素受体及其下游效应分子CARD9表达与临床病理特征和预后的关系

目的探究直肠癌患者的血清C型凝集素受体(CLR)及其下游效应分子胱天蛋白酶募集域蛋白9(CARD9)表达与临床病理特征和预后的关系。方法选择2018年3月至2020年3月国药同煤总医院收治的98例直肠癌患者作为研究对象(设为观察组),另选择45名同期在该院体检的健康志愿者设为对照组。采用ELISA法检测血清可溶性树突状细胞特异性细胞间黏附分子3结合非整合素(sDC-SIGN)和CARD9表达水平,并分析其与直肠癌患者临床病理特征的关系。采用Pearson相关性分析探讨血清sDC-SIGN与CARD9表达的关系。入组直肠癌患者术后均随访3年,计算3年总生存(OS)率。采用Kaplan-Meier法绘制生存曲线,分析血清sDC-SIGN和CARD9表达与直肠癌患者预后的关系。采用多因素Cox回归分析探讨直肠癌患者预后的影响因素。采用ROC曲线分析血清sDC-SIGN和CARD9对直肠癌患者术后生存情况的预测价值。结果与对照组相比,观察组的血清sDC-SIGN表达水平降低,血清CARD9表达水平升高,2组的差异均有统计学意义(P均<0.05)。Pearson相关性分析结果显示,直肠癌患者的血清sDC-SIGN与CARD9表达呈显著负相关(r=-0.743,P<0.05)。与无淋巴结转移的直肠癌患者相比,有淋巴结转移的患者的血清sDC-SIGN表达水平显著降低,而血清CARD9表达水平显著升高,差异均有统计学意义(P均<0.05)。sDC-SIGN低表达组的3年OS率显著低于sDC-SIGN高表达组,CARD9高表达组的3年OS率显著低于CARD9低表达组,差异均有统计学意义(P均<0.05)。多因素Cox回归分析结果显示,分化程度、TNM分期、淋巴结转移、血清sDC-SIGN和CARD9均是影响直肠癌患者预后的独立危险因素(P均<0.05)。血清sDC-SIGN和CARD9联合预测直肠癌患者术后3年生存情况的ROC曲线下面积(AUC)为0.896,均大于两者单独预测的AUC(P均<0.05)。结论血清sDC-SIGN和CARD9表达水平均与直肠癌发生密切相关,且血清sDC-SIGN低表达及血清CARD9高表达均提示直肠癌患者术后预后较差,其可能成为直肠癌诊断和预后预测的新血清学标志物。

Physiological effects of amyloid precursor protein and its derivatives on neural stem cell biology and signaling pathways involved

The pathological implication of amyloid precursor protein(APP)in Alzheimer’s disease has been widely documented due to its involvement in the generation of amyloid-β peptide.However,the physiological functions of APP are still poorly understood.APP is considered a multimodal protein due to its role in a wide variety of processes,both in the embryo and in the adult brain.Specifically,APP seems to play a key role in the proliferation,differentiation and maturation of neural stem cells.In addition,APP can be processed through two canonical processing pathways,generating different functionally active fragments:soluble APP-α,soluble APP-β,amyloid-β peptide and the APP intracellular C-terminal domain.These fragments also appear to modulate various functions in neural stem cells,including the processes of proliferation,neurogenesis,gliogenesis or cell death.However,the molecular mechanisms involved in these effects are still unclear.In this review,we summarize the physiological functions of APP and its main proteolytic derivatives in neural stem cells,as well as the possible signaling pathways that could be implicated in these effects.The knowledge of these functions and signaling pathways involved in the onset or during the development of Alzheimer’s disease is essential to advance the understanding of the pathogenesis of Alzheimer’s disease,and in the search for potential therapeutic targets.

Nickel uptake and intracellular localization in Cupriavidus pauculus KPS 201, native to ultramafic ecosystem

The nickel-resistant bacterium, Cupriavidus pauculus KPS 201 was isolated from the rhizosphere of Rinorea bengalensis (Wall.) O. K. endemic to metal-percolated ultramafic ecosystem of Andaman, India. This study investigates nature of Ni resistance, growth associated uptake and localization of Ni in cellular compartments of KPS 201. Growth kinetics of C. pauculus KPS 201 exhibited a typical inducible Ni resistance in Ni-supplemented (1.0-10.0 mM) Tris-minimal medium. The Ni-induced cells showed a high degree of Ni resistance and accumulated a maximum of 29.3 μM Ni/g protein after 48 h of growth in 5 mM Ni. The accumulated Ni was preferentially retained (90.6%) in the periplasm and was associated with the expression of two periplasmic proteins (74 and 66 kDa) under Ni-induced condition. Inducible nickel resistance in C. pauculus KPS 201 may possibly be due to extracytoplasmic binding and accumulation coupled with expression of specific periplasmic proteins. These findings will provide an insight in understanding metal-microbe interaction in geogenous environments and their exploitation in bioremediation of heavy metal pollutants.

Cloning and characterization of a novel gene encoding a putative seven-span transmembrane protein localized in endoplasmic reticulum

Objective: To clone and analyze the structure of a novel gene, named EST 1 (endoplasmic reticulum localized seven span transmembrane protein 1) and to analyze the expression pattern and intracellular location of EST 1. Methods: The cDNA library was screened to isolate novel cDNA fragment. The structure of novel gene was analysed by computer software. Expression of EST 1 was analyzed by dot blot and Northern blotting. Intracellular localization was observed after EST 1 enhanced green fluorescence protein (EGFP) fusion gene was transfected into mammalian cells. Results: The full length cDNA of mouse EST 1 was 1 802 bp, with a 1 293 bp open reading frame encoding 431 amino acids. It was predicated that protein encoded by EST 1 contained a signal peptide sequence at the N terminus, seven putative transmembrane domains, and an ER retaining signal at the C terminus. EST 1 EGFP fusion protein showed an ER like intracellular distribution in mammalian cells. Expression pattern analysis showed that EST 1 is expressed in all tissues examined. Conclusion: EST 1 is encoding a putative seven span transmembrane protein localized in endoplasmic reticulum. EST 1 was expressed in all tissues examined, suggesting an essential function of EST 1 in cells.

Protein@PP-Zn nanocomplex assembled by coordination of zinc ions used for intracellular protein delivery

In recent years,intracellular delivery of protein drugs has attracted great attention,and polymer-based systems have been extensively exploited to develop efficient and safe carriers.However,efficient intracellular delivery of protein drugs remains a challenge because of the cell membrane barrier and endosome entrapment.Herein,we report a protein@PP-Zn nanocomplex,which consists of an imidazole-containing block polymer poly(ethylene glycol)-block-poly(β-amino ester)(PEG-b-PAE(Im),PP),zinc ions,and protein drugs,for efficient intracellular protein delivery.PEG-b-PAE(Im)could conjugate proteins via the bridging effect of zinc ions which simultaneously coordinate with imidazole groups on polymer and electron donor groups,such as imidazole and primary amine groups,on protein to improve the loading stability of proteins.Under a slightly acidic environment near cancer cells,the protonation of PAE(Im)backbone increases the positive charge density of the nanocomplex and promotes endocytosis.While under a more acidic environment in endosomes,further protonation of imidazole groups leads to the disintegration of the nanocomplex and the breakdown of endosomes because of the proton sponge effect.Finally,protein is released into the cytoplasm.With the assistance of the nanocomplex,proteins with different sizes and isoelectric points are effectively delivered into cells.This work provides a stable,efficient and universal strategy for intracellular protein delivery.

同向马达蛋白在细胞内的运输

细胞内物质的主动运输主要通过马达蛋白这一特殊的纳米机器来完成。在运输过程中,马达蛋白之间的协作性显著影响着距离、速度等重要的运输特性。为了理解这一机制,我们结合Gillespie模拟和理论推导,解释了单马达蛋白的力学特性如何影响了多个马达蛋白之间的协作性,进而对运输距离进行调节。我们建立了一个深度学习模型来帮助快速获得马达蛋白的运动参数。我们的结果揭示了同向马达蛋白在细胞内运输的物理本质。

黄芪多糖对小鼠免疫细胞信号转导相关分子的影响

为探讨黄芪多糖(APS)免疫调节的作用机理,观察了黄芪多糖对小鼠腹腔巨噬细胞一氧化氮(NO)生成、对体外培养的小鼠脾脏淋巴细胞内游离钙离子水平和蛋白激酶C(PKC)活性的影响。结果显示黄芪多糖能明显促进小白鼠腹腔巨噬细胞NO生成,显著升高小鼠脾淋巴细胞内游离钙离子的水平,引起细胞PKC活性明显升高。提示黄芪多糖通过NO介导信号传递通路,调节脾淋巴细胞内游离钙离子的浓度,升高细胞蛋白激酶活性而影响机体免疫细胞的信号转导,发挥其免疫调节作用。