Study of protein kinase C antagonists on cortical granule exocytosis and cell-cycle resumption in fertilized mouse eggs

Although pharmacological agonists of protein kinase C (PKC) stimulate some events of mammalian egg activation, including cortical granule (CG) exocytosis, it is not known if these events are dependent on PKC activation during the normal process of fertilization. In order to examine the potential role of PKC in CG exocytosis, this study investigated whether PKC agonists faithfully mimic CG release and whether PKC antagonists block fertilization-induced CG release in mature mouse eggs. Phorbol ester (TPA, 2.5 ng/ml) treatment resulted in an atypical pattern of CG release in which there was a greater net loss of CGs in the equatorial region of the egg than in the region opposite the spindle. This pattern also was in contrast to that during fertilization, in which CG release occurred randomly throughout the cortex. Fertilization experiments utilized two different PKC inhibitors, bisindolyl-maleimide (5 μM) and chelerytherine (0.8 μM), targeted to both the “conserved” substrate and ATP binding domains of PKC. Simultaneous use of both inhibitors at maximal concentrations (compatible with fertilization and above their IC₅₀S) resulted in no detectable inhibition of CG release in treated fertilized eggs compared to controls. In addition, no inhibition of anaphase onset was observed in treated fertilized eggs. Activity of the inhibitors was verified by demonstrating that they blocked the induction of CG loss by TPA. Moreover, 1 μM staurosporine, a potent but less specific antagonist of PKC, also did not block CG loss, whereas the metaphase-anaphase transition was temporarily inhibited. The results indicate that TPA does not faithfully mimic CG release in fertilized eggs, that a role for PKC in CG release at fertilization remains to be established, and that other calcium-dependent effectors may be involved in CG exocytosis. Mol Reprod Dev 46:216–226, 1997. © 1997 Wiley-Liss, Inc.     

An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function

Stress granules (SG) are membrane‐less compartments involved in regulating mRNAs during stress. Aberrant forms of SGs have been implicated in age‐related diseases, such as amyotrophic lateral sclerosis (ALS), but the molecular events triggering their formation are still unknown. Here, we find that misfolded proteins, such as ALS‐linked variants of SOD1, specifically accumulate and aggregate within SGs in human cells. This decreases the dynamics of SGs, changes SG composition, and triggers an aberrant liquid‐to‐solid transition of in vitro reconstituted compartments. We show that chaperone recruitment prevents the formation of aberrant SGs and promotes SG disassembly when the stress subsides. Moreover, we identify a backup system for SG clearance, which involves transport of aberrant SGs to the aggresome and their degradation by autophagy. Thus, cells employ a system of SG quality control to prevent accumulation of misfolded proteins and maintain the dynamic state of SGs, which may have relevance for ALS and related diseases.     

八正颗粒与左氧氟沙星对单纯性急性尿路感染患者血清PCT及IL-8水平的影响及其临床疗效

目的:探讨八正颗粒联合左氧氟沙星对单纯性急性尿路感染血清降钙素原(PCT)、白细胞介素-8(IL-8)水平及临床疗效的影响。方法:选取我院泌尿科收治的单纯性急性尿路感染72例,随机分为对照组和观察组,每组36例。对照组患者予以左氧氟沙星治疗,观察组患者在对照组基础上予以八正颗粒治疗。观察并记录两组间血清降钙素原(PCT)、白细胞介素-8(IL-8)水平、症状改善时间、临床疗效及不良反应的发生情况。结果:对照组治疗有效率为72.22%,显著低于观察组(91.67%),差异具有统计学意义(P0.05);观察组尿路疼痛、腰痛、尿频、尿急等症状改善所需的时间均明显短于对照组,治疗后血清PCT、IL-8水平显著低于对照组,差异均具有统计学意义(P0.05)。结论:八正颗粒联合左氧氟沙星治疗单纯性急性尿路感染的临床效果较单用左氧氟沙星治疗更好,且能有效降低患者血清PCT、IL-8水平。     

二甲双胍联合津力达颗粒治疗妊娠期糖尿病的临床效果及对患者血清VEGF、APN、Hcy水平的影响

目的:探讨二甲双胍联合津力达颗粒治疗妊娠期糖尿病的临床效果及对患者血清血管内皮生长因子(VEGF)、脂联素(APN)、同型半胱氨酸(Hcy)水平的影响。方法:选择2014年7月至2016年7月我院接诊的94例妊娠期糖尿病患者并通过随机数表法分为观察组(n=47)和对照组(n=47)。对照组使用二甲双胍治疗,观察组在对照组的基础上联合津力达颗粒治疗,均治疗至胎儿娩出。比较两组治疗前后血糖、血脂及血清VEGF、APN和Hcy水平的变化及产妇并发症和新生儿不良结局的发生情况。结果:与治疗前比较,两组治疗后血糖、血脂指标均显著改善(P0.05),观察组空腹血糖(FBG)、餐后2 h血糖(2h PG)、糖化血红蛋白(Hb A1c)、总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)水平均明显低于对照组,血清高密度脂蛋白胆固醇(HDL-C)水平明显高于对照组(P0.05)。两组治疗后血清VEGF、APN、Hcy水平较治疗前均显著改善(P0.05),且观察组血清VEGF、Hcy均明显低于对照组,血清APN水平明显高于对照组(P0.05)。观察组妊娠期高血压、羊水过多、剖宫产、早产、巨大儿、新生儿黄疸、新生儿呼吸窘迫的发生率均明显低于对照组(P0.05)。结论:二甲双胍联合津力达颗粒治疗妊娠期糖尿病的临床效果显著,可有效控制血糖、血脂水平,降低不良母婴结局的发生率,可能与其有效调节血清VEGF、APN、Hcy水平有关。     

SND1蛋白与HuR蛋白的相互结合作用

人源SND1（staphylococcal nuclease domain containing 1）蛋白由N端的SN（staphylococcal nucleases）结构域和C端的TSN (Tudor SN5) 结构域组成,其中SN结构域又包含SN1～SN4四个重复的功能片段. 本课题组前期研究结果表明,SND1蛋白可以通过SN结构域与G3BP（Ras GAP SH3 domain binding protein）蛋白相互结合,共同参与细胞应激颗粒（stress granules,SGs）的形成. SGs是真核细胞在受到氧化应激、病毒感染等外界刺激时在胞浆内形成的与RNA代谢相关的颗粒状结构. 对于SGs的成分鉴定及相互作用的分析一直是学者们研究的热点. 本研究中,免疫共沉淀实验结果表明,以抗SND1抗体可以共沉淀出HeLa细胞内另一个重要的应激相关人类抗原R（human antigen R,HuR）蛋白. 另外,利用脂质体转染法将pcDNA3 FLAG HuR重组质粒瞬时转染入HeLa细胞,成功过表达外源性的FLAG HuR融合蛋白,再以抗FLAG标签抗体又可以反向共沉淀出内源性SND1蛋白,证明SND1与HuR之间存在蛋白质间的相互结合作用. 细胞免疫荧光实验结果表明,当给予HeLa细胞05 mmol/L亚砷酸钠氧化应激时,SND1与HuR蛋白共同定位于胞浆中的SGs结构中. GST pulldown实验结果进一步表明截短的SN结构域可以结合HuR蛋白,其中以SN1功能片段的结合能力最强,表明SND1蛋白是通过SN结构域与HuR蛋白形成应激复合物,参与SGs的胞浆组装.并不定位于SGs的TSN结构域亦可结合HuR蛋白,提示SND1 HuR的蛋白相互作用可能并不局限于SGs,具有其它方面的功能意义.     

The plant sHSP superfamily: five new members in Arabidopsis thaliana with unexpected properties

The small heat shock proteins (sHsps), which are ubiquitous stress proteins proposed to act as chaperones, are encoded by an unusually complex gene family in plants. Plant sHsps are classified into different subfamilies according to amino acid sequence similarity and localization to distinct subcellular compartments. In the whole Arabidopsis thaliana genome, 19 genes were annotated to encode sHsps, of which 14 belong to previously defined plant sHsp families. In this paper, we report studies of the five additional sHsp genes in A. thaliana, which can now be shown to represent evolutionarily distinct sHsp subfamilies also found in other plant species. While two of these five sHsps show expression patterns typical of the other 14 genes, three have unusual tissue specific and developmental profiles and do not respond to heat induction. Analysis of intracellular targeting indicates that one sHsp represents a new class of mitochondrion-targeted sHsps, while the others are cytosolic/nuclear, some of which may cooperate with other sHsps in formation of heat stress granules. Three of the five new proteins were purified and tested for chaperone activity in vitro. Altogether, these studies complete our basic understanding of the sHsp chaperone family in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Toxoplasma gondii uses unusual sorting mechanisms to deliver transmembrane proteins into the host-cell vacuole

A critical step in infection by the apicomplexan parasite Toxoplasma gondii is the formation of a membrane-bound compartment within which the parasite proliferates. This process relies on a set of secretory organelles that discharge their contents into the host cell upon invasion. Among these organelles, the dense granules are specialized in the export of transmembrane (TM) GRA proteins, which are major components of the mature parasitophorous vacuole (PV) membrane. How eukaryotic pathogens export and sort membrane-bound proteins destined for the host cell is still poorly understood at the mechanistic level. In this study, we show that soluble trafficking of the PV-targeted GRA5 TM protein is parasite specific: when expressed in mammalian cells, GRA5 is targeted to the plasma membrane and behaves as an integral membrane protein with a type I toplogy. We also demonstrate the dual role of the GRA5 N-terminal ectodomain, which is sufficient to prevent membrane integration within the parasite and is essential for both sorting and post-secretory membrane insertion into the vacuolar membrane. These results contrast with the general rule that states that information contained within the cytoplasmic tail and/or the TM domain of integral membrane proteins dictates their cellular localization. They also highlight the diversity of sorting mechanisms that leads to the specialization of secretory processes uniquely adapted to intracellular parasitism.     

Characterization of Ayu17-449 gene expression and resultant kidney pathology in a knockout mouse model

The gene trap technique is a powerful approach for characterizing and mutating genes in the mouse. We used this method to identify a mouse gene of unknown function and to establish a mutant mouse line. We subsequently identified one gene, denoted Ayu17-449, on mouse chromosome 3 that comprised 14 exons encoding 1920 amino acids with a granin motif in its N-terminal sequence. In adult mice, this gene was highly expressed in the brain, heart, lung, muscle, stomach, and kidney. The insertion of a trap vector into the second intron of this gene resulted in the null mutation. Homozygous mice for these mutation died by 1 day after birth. Mutant mice showed a loss of acidic granules in the proximal convoluted tubules of the kidney. Our data demonstrates that Ayu17-449 is important for mouse survival.     

Understanding the properties of aerobic sludge granules as hydrogels

Aerobic sludge granules are larger, denser microbial aggregates than activated sludge flocs with a smoother and more regular surface, which facilitates greater wastewater treatment intensity. Factors important in their growth are still poorly understood, which is an impediment to the construction and operation of full-scale aerobic sludge granule processes. Data in this article obtained with granules treating an abattoir wastewater provide evidence that aerobic sludge granules are hydrogels. The results also demonstrate a method for characterizing macromolecular associations. The rheological profile of these granules was found to be analogous with that of typical polymer gels. Water uptake or swelling reflects an equilibrium between granule elastic modulus and osmotic pressure, whereby uptake is increased by reducing solute concentration or the elastic modulus. A weakening of the extracellular polymeric substance (EPS) matrix as demonstrated with mechanical spectroscopy was induced by several environmental factors including temperature, pH and ionic strength. Uniform and elastic deformation was observed at low strain. Enzymatic degradation studies indicate that proteins and alpha-polysaccharides were the major granule structural materials. The aerobic sludge granules in the current study were therefore protein-polysaccharide composite physical hydrogels. While aerobic sludge granules treating an abattoir wastewater are used as a case study, many of the fundamental principles detailed here are relevant to other granulation processes. The paradigm established in this study can potentially be applied to better understand the formation of aerobic sludge granules and thus overcome a hurdle in the acceptance of aerobic sludge granulation as an alternative to more traditional wastewater treatment processes.     

黄蜂胃部表皮黄色颗粒的微观形态与发育(英文)

东方大黄蜂（胡蜂）Vespa orientalis（膜翅目Vespinae属）的胃部表皮黄色颗粒位于黄色条纹区域。从黄色颗粒产生至在这一区域扩散,这个过程就是胃部黄颜色形成的过程。用几种电子显微镜研究了黄色颗粒的微观形态和发育过程。结果显示黄色颗粒由20—25 µm厚的一层组成,包括总表皮在内厚度约为40—45 µm。从上面看,在上述的区域能看到许多直径大约为0.5 µm周边光感受器细胞(PPC)。在每个黄色颗粒,能观察到一个肌样包膜,它位于一个由9根原纤维组装而成的圆圈内部。黄色颗粒成熟的过程包含通过导管的渗透增加初生黄色粒子数量,这些初生粒子又发育或增生成次生粒子,次生粒子逐渐布满整个区域。在这些黄色粒子层内可产生类似于肝的功能活动。