ACTIVE CALCIUM TRANSPORT IN PLASMA MEMBRANE VESICLES FROM DEVELOPING COTYLEDONS OF COMMON BEAN

Plasma membrane vesicles were prepared from the developing cotyledons of common beau (Phaseolus vulgaris L cv Diyundou) by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes for other membranes.The putative plasma membrane fraction was minimally contaminated by membranes Other than plasma membrane and hence was of high purity. It exhibited a Ca2+dependent ATPase activity, which was inhibited by 1μ mol/L EB and promoted by calcium ionophore A23187. Such an activity was responsible for the observed ATPdependent 45Ca2+ uptake into inside-out plasma membrane vesicles. This process was stimulated by 0.6μmol/L CaM and 20μmol/L IAA but inhibited by 2μmol/L ABA and abolished by A23187. Possible role of cytoplasmic Ca2+ in mediating phytohormones activity is discussed.

Serum outperforms plasma in small extracellular vesicle microRNA biomarker studies of adenocarcinoma of the esophagus

BACKGROUND Circulating microRNAs(miRNAs)are potential biomarkers for many diseases.However,they can originate from non-disease specific sources,such as blood cells,and compromise the investigations for miRNA biomarkers.While small extracellular vesicles(sEVs)have been suggested to provide a purer source of circulating miRNAs for biomarkers discovery,the most suitable blood sample for sEV miRNA biomarker studies has not been defined.AIM To compare the mi RNA profiles between matched serum and plasma s EV preparations to determine their suitability for biomarker studies.METHODS Matched serum and plasma samples were obtained from 10 healthy controls and10 patients with esophageal adenocarcinoma.s EV isolates were prepared from serum and plasma using Exo Quick TM and quantified using Nano Sight.RNA was extracted from s EV preparations with the mi RNeasy Serum/Plasma kit and profiled using the Taqman Openarray q PCR.The overall mi RNA content and theexpression of specific mi RNAs of reported vesicular and non-vesicular origins were compared between serum and plasma s EV preparations.The diagnostic performance of a previously identified multi-mi RNA biomarker panel for esophageal adenocarcinoma was also compared.RESULTS The overall mi RNA content was higher in plasma s EV preparations(480 mi RNAs)and contained 97.5%of the mi RNAs found in the serum s EV preparations(412 mi RNAs).The expression of commonly expressed mi RNAs was highly correlated(Spearman’s R=0.87,P<0.0001)between the plasma and serum s EV preparations,but was consistently higher in the plasma s EV preparations.Specific blood-cell mi RNAs(hsa-mi R-223-3 p,hsa-mi R-451 a,mi R-19 b-3 p,hsa-mi R-17-5 p,hsa-mi R-30 b-5 p,hsa-mi R-106 a-5 p,hsa-mi R-150-5 p and hsa-mi R-92 a-3 p)were expressed at 2.7 to 9.6 fold higher levels in the plasma s EV preparations compared to serum s EV preparations(P<0.05).In plasma s EV preparations,the percentage of protein-associated mi RNAs expressed at relatively higher levels(Ct 20-25)was greater than serum s EV preparations(50%vs 31%).While the percentage of vesicle-associated mi RNAs expressed at relatively higher levels was greater in the serum s EV preparations than plasma s EV preparations(70%vs 44%).A 5-mi RNA biomarker panel produced a higher cross validated accuracy for discriminating patients with esophageal adenocarcinoma from healthy controls using serum s EV preparations compared with plasma s EV preparations(AUROC 0.80 vs 0.54,P<0.05).CONCLUSION Although plasma s EV preparations contained more mi RNAs than serum s EV preparations,they also contained more mi RNAs from non-vesicle origins.Serum appears to be more suitable than plasma for s EV mi RNAs biomarkers studies.

Standardization of Sampling for Isolation of Exosome-Like Small-Extracellular Vesicles from Peripheral Blood from Reproductive-Aged Women

Exosome-like small-extracellular vesicles (sEVs) are extracellular vesicles that act in intercellular communication and are involved in several biologic and pathologic processes. While sEVs increase the stability of their cargo molecules, there is still a need for standardization of sampling and isolation of these microvesicles. We aimed to determine the best sampling method for isolation of sEVs from peripheral blood from reproductive-aged women. Material and Methods: We included samples of plasma from our biobank collected in 2014 by venipuncture in heparin tubes and stored at -80°C. We also included blood samples collected in heparin tubes and Ethylenediamine tetraacetic acid (EDTA) tubes and stored at -80°C for one to two weeks prior processing. All blood samples were collected from the same nine reproductive-aged female volunteers. sEVs were isolated from plasma by ultracentrifugation and filtration and indirectly quantified using Pierce BCA Protein Assay kit. Transmission electron microscopy (TEM) and Nano Tracking Analysis (NTA) were performed to confirm the isolation of sEVs. Results and Discussion: TEM and NTA confirmed the isolation of sEVs. Protein concentration of short-time stored heparin samples was not statistically different from long-time stored heparin samples (1847.2 ± 651.4 vs. 2363.2 ± 1025.1, p = 0.14). There was no difference between heparin and EDTA plasma samples recently collected (2363.2 ± 1025.1 vs. 2044.8 ± 653.2, p = 0.44). In conclusion, blood samples may be collected using heparin or EDTA for isolation of sEVs. Long-time stored plasma samples maintain sEVs integrity and may be used, especially in comparative studies.

富血小板血浆来源的血小板细胞外囊泡分离技术与应用

背景:血小板细胞外囊泡是循环中最丰富的囊泡,富含丰富的生物活性分子、遗传物质及蛋白质等信息分子,参与细胞通讯和物质交换,不仅具有良好的促凝活性,还具有促进组织修复和再生的巨大潜力,在再生医学具有广泛的应用前景。目的:从血小板细胞外囊泡的分泌机制、在再生医学领域的应用及临床转化的限制因素进行阐述,为血小板细胞外囊泡的临床转化提供一些理论支撑。方法:应用计算机检索2005年1月至2023年8月PubMed数据库与血小板细胞外囊泡有关的文章,英文检索词为“platelet-derived,plateletrich plasma,extracellular vesicles,isolated,microvesicles exosomes,applications”,最后纳入符合主题标准的文献62篇进行综述分析。结果与结论:(1)活化的血小板细胞外囊泡可产生血小板微囊泡和血小板外泌体两种类型的囊泡,前者的分泌可能与肌动蛋白细胞骨架的不对称性相关,后者的分泌可能与H^(+)-ATP酶的调节相关。(2)血小板细胞外囊泡是血小板浓缩物和血小板本身的潜在效应物,可能通过促进血管生成、影响细胞行为、促凝与止血以及发挥炎症作用等几方面影响组织再生。(3)血小板细胞外囊泡在组织损伤、肌肉再生、软骨再生、骨关节炎等再生医学领域已有临床前报道,作为伤口愈合的潜在治疗方法已有临床试验数据,但血小板细胞外囊泡的分离方法、样本来源以及激活剂的种类等因素限制了血小板细胞外囊泡在再生医学领域的临床转化。(4)未来血小板细胞外囊泡可能成为再生医学中替代富血小板血浆的无细胞疗法,其临床转化还需要积极寻找鉴别血小板细胞外囊泡和血小板外泌体的特异性标志物,在激活剂刺激血小板细胞外囊泡产生的机制以及血小板细胞外囊泡的最佳收集方式、最佳储存方法、保质期限、临床应用的推荐剂量和最佳临床适应证还需要继续深入研究。

MicroRNAs as potential diagnostic biomarkers for bipolar disorder

Abnormal expression of microRNAs is connected to brain development and disease and could provide novel biomarkers for the diagnosis and prognosis of bipolar disorder. We performed a PubMed search for microRNA biomarkers in bipolar disorder and found 18 original research articles on studies performed with human patients and published from January 2011 to June 2023. These studies included microRNA profiling in bloodand brain-based materials. From the studies that had validated the preliminary findings,potential candidate biomarkers for bipolar disorder in adults could be miR-140-3p,-30d-5p,-330-5p,-378a-5p,-21-3p,-330-3p,-345-5p in whole blood, miR-19b-3p,-1180-3p,-125a-5p, let-7e-5p in blood plasma, and miR-7-5p,-23b-5p,-142-3p,-221-5p,-370-3p in the blood serum. Two of the studies had investigated the changes in microRNA expression of patients with bipolar disorder receiving treatment. One showed a significant increase in plasma miR-134 compared to baseline after 4 weeks of treatment which included typical antipsychotics, atypical antipsychotics, and benzodiazepines. The other study had assessed the effects of prescribed medications which included neurotransmitter receptorsite binders(drug class B) and sedatives, hypnotics, anticonvulsants, and analgesics(drug class C) on microRNA results. The combined effects of the two drug classes increased the significance of the results for miR-219 and-29c with miR-30e-3p and-526b* acquiring significance. MicroRNAs were tested to see if they could serve as biomarkers of bipolar disorder at different clinical states of mania, depression, and euthymia. One study showed that upregulation in whole blood of miR-9-5p,-29a-3p,-106a-5p,-106b-5p,-107,-125a-3p,-125b-5p and of miR-107,-125a-3p occurred in manic and euthymic patients compared to controls, respectively, and that upregulation of miR-106a-5p,-107 was found for manic compared to euthymic patients. In two other studies using blood plasma,downregulation of miR-134 was observed in manic patients compared to controls, and dysregulation of miR-134,-152,-607,-633,-652,-155 occurred in euthymic patients compared to controls. Finally, microRNAs such as miR-34a,-34b,-34c,-137, and-140-3p,-21-3p,-30d-5p,-330-5p,-378a-5p,-134,-19b-3p were shown to have diagnostic potential in distinguishing bipolar disorder patients from schizophrenia or major depressive disorder patients, respectively. Further studies are warranted with adolescents and young adults having bipolar disorder and consideration should be given to using animal models of the disorder to investigate the effects of suppressing or overexpressing specific microRNAs.

MicroRNAs as potential biomarkers for diagnosis of post-traumatic stress disorder

Post-traumatic stress disorder is a mental disorder caused by exposure to severe traumatic life events.Currently,there are no validated biomarkers or laboratory tests that can distinguish between trauma survivors with and without post-traumatic stress disorder.In addition,the heterogeneity of clinical presentations of post-traumatic stress disorder and the overlap of symptoms with other conditions can lead to misdiagnosis and inappropriate treatment.Evidence suggests that this condition is a multisystem disorder that affects many biological systems,raising the possibility that peripheral markers of disease may be used to diagnose post-traumatic stress disorder.We performed a PubMed search for microRNAs(miRNAs)in post-traumatic stress disorder(PTSD)that could serve as diagnostic biomarkers and found 18 original research articles on studies performed with human patients and published January 2012 to December 2023.These included four studies with whole blood,seven with peripheral blood mononuclear cells,four with plasma extracellular vesicles/exosomes,and one with serum exosomes.One of these studies had also used whole plasma.Two studies were excluded as they did not involve microRNA biomarkers.Most of the studies had collected samples from adult male Veterans who had returned from deployment and been exposed to combat,and only two were from recently traumatized adult subjects.In measuring miRNA expression levels,many of the studies had used microarray miRNA analysis,miRNA Seq analysis,or NanoString panels.Only six studies had used real time polymerase chain reaction assay to determine/validate miRNA expression in PTSD subjects compared to controls.The miRNAs that were found/validated in these studies may be considered as potential candidate biomarkers for PTSD and include miR-3130-5p in whole blood;miR-193a-5p,-7113-5p,-125a,-181c,and-671-5p in peripheral blood mononuclear cells;miR-10b-5p,-203a-3p,-4488,-502-3p,-874-3p,-5100,and-7641 in plasma extracellular vesicles/exosomes;and miR-18a-3p and-7-1-5p in blood plasma.Several important limitations identified in the studies need to be taken into account in future studies.Further studies are warranted with war veterans and recently traumatized children,adolescents,and adults having PTSD and use of animal models subjected to various stressors and the effects of suppressing or overexpressing specific microRNAs.

1%富血小板血浆联合骨髓间充质干细胞促进周围神经损伤的修复

背景:富血小板血浆已被证明能够增强间充质干细胞活性并具有促血管再生能力。细胞外囊泡是间充质干细胞发挥作用的关键介质之一,但目前还不清楚富血小板血浆是否能影响细胞外囊泡的功能。目的:探索富血小板血浆对骨髓间充质干细胞分泌的细胞外囊泡功能的影响,验证是否能够将富血小板血浆用作一种刺激剂来改善骨髓间充质干细胞修复周围神经损伤的效果。方法:体外实验方面,在一般条件下和含有1%富血小板血浆的条件下培养骨髓间充质干细胞,用超速离心法分离骨髓间充质干细胞分泌的细胞外囊泡,前者命名为EVs-nor,后者命名为EVs-prp。将细胞外囊泡用于干预施万细胞,通过EdU、蛋白印迹、qPCR、光学显微镜拍照等分析施万细胞修复性重编程相关特征,包括细胞增殖、c-Jun蛋白表达、重编程相关基因表达、细胞形态等。体内实验方面,构建大鼠坐骨神经断伤模型,然后利用甲壳素神经套管将骨髓间充质干细胞单独或连同1%富血小板血浆植入损伤神经,术后8周评估组织学和功能学恢复情况,评估指标包括再生神经纤维密度、腓肠肌肌肉湿质量、坐骨神经功能指数等。结果与结论:(1)相比于EVs-nor,EVs-prp刺激后的施万细胞增殖能力更强,c-Jun基因表达水平更高,GDNF表达上调,细胞形态更长,表明EVs-prp比EVs-nor有更强的刺激施万细胞重编程的能力;(2)坐骨神经损伤动物实验结果显示,相比于单独植入骨髓间充质干细胞或单独植入富血小板血浆,将骨髓间充质干细胞联合富血小板血浆一起植入的修复效果最好,具体体现在该组的再生神经纤维密度、腓肠肌肌肉湿质量、坐骨神经功能指数等指标均显著优于其他组;(3)这些实验结果显示,富血小板血浆可以改善骨髓间充质干细胞来源细胞外囊泡的功能,并且可以作为一种实用、易得的制剂与骨髓间充质干细胞协同促进周围神经损伤修复。

Autism spectrum disorder:difficulties in diagnosis and microRNA biomarkers

We performed a PubMed search for microRNAs in autism spectrum disorder that could serve as diagnostic biomarkers in patients and selected 17 articles published from January 2008 to December 2023,of which 4 studies were performed with whole blood,4 with blood plasma,5 with blood serum,1 with serum neural cell adhesion molecule L1-captured extracellular vesicles,1 with blood cells,and 2 with peripheral blood mononuclear cells.Most of the studies involved children and the study cohorts were largely males.Many of the studies had performed microRNA sequencing or quantitative polymerase chain reaction assays to measure microRNA expression.Only five studies had used real-time polymerase chain reaction assay to validate microRNA expression in autism spectrum disorder subjects compared to controls.The microRNAs that were validated in these studies may be considered as potential candidate biomarkers for autism spectrum disorder and include miR-500a-5p,-197-5p,-424-5p,-664a-3p,-365a-3p,-619-5p,-664a-3p,-3135a,-328-3p,and-500a-5p in blood plasma and miR-151a-3p,-181b-5p,-320a,-328,-433,-489,-572,-663a,-101-3p,-106b-5p,-19b-3p,-195-5p,and-130a-3p in blood serum of children,and miR-15b-5p and-6126 in whole blood of adults.Several important limitations were identified in the studies reviewed,and need to be taken into account in future studies.Further studies are warranted with children and adults having different levels of autism spectrum disorder severity and consideration should be given to using animal models of autism spectrum disorder to investigate the effects of suppressing or overexpressing specific microRNAs as a novel therapy.

Characterization of Exosomes in Plasma of Patients with Breast, Ovarian, Prostate, Hepatic, Gastric, Colon, and Pancreatic Cancers

Detection of circulating tumor-specific DNA, RNA or proteins can be difficult due to relative scarcity. Exosomes are extracellular vesicles, 30 - 150 nm in diameter derived from fusion of multivesicular bodies with the plasma membrane. They are composed of a lipid bilayer membrane and contain proteins, mRNA and miRNA. Exosomes are secreted by multiple cell types, including cancer cells. However, there is a relative lack of information concerning the contents of exosomes secreted by various tumor cell types. To examine exosomes in cancer, we collected blood plasma samples from patients with breast, ovarian, prostate, hepatic, gastric, colon, and pancreatic cancers. Exosomes were isolated from plasma and confirmed by AchE assay, transmission electron microscopy and expression of the CD63 exosomal marker. Expression of AFP, CA724, CA153, CEA, CA125, CA199 and PSA antigens were determined using an automated electro-chemiluminescence assay. Expression of the tumor-related chaperone protein, mortalin, was determined by Western blot analysis. Levels of exosome secretion were variable among the different tumor types. Both exosome levels and mortalin expression within tumor cell exosomes were higher than in healthy donors, except in pancreatic carcinoma, where exosomes were elevated but mortalin expression was not significantly different from healthy donors. Exosomes provide unique opportunities for the enrichment of tumor-specific materials and may be useful as biomarkers and possibly as tools of cancer therapies. Mortalin, which has been linked to cell proliferation and induction of epithelial-mesenchymal transition of cancer cells, may be useful as a prognostic biomarker and as a possible therapeutic target.

富血小板血浆来源外泌体治疗膝骨关节炎的机制与作用

背景:骨关节炎是一种常见的关节退行性疾病,再生疗法促进膝骨关节炎软骨修复一直是研究热点,其中富血小板血浆来源外泌体可能是一种新的再生治疗方式。目的:综述目前文献,总结富血小板血浆来源外泌体在膝骨关节炎中的应用、作用以及潜在机制。方法:以“platelet rich plasma,extracellular vesicles,exosomes,knee osteoarthritis”为英文关键词、以“富血小板血浆,细胞外囊泡,外泌体,膝骨关节炎”为中文关键词分别在Embase、Medline、Cochrane Central Register of Controlled Trials、PubMed、万方数据库及中国知网检索。初检得到1007篇文献,按纳入、排除标准最终共入选65篇文献进行综述。结果与结论:①富血小板血浆来源外泌体的制备涉及富血小板血浆的制备、激活以及使用试剂盒或者超高速离心法获取外泌体,鉴定方法包括标志物表达检测、形态学观察、颗粒跟踪分析等;②富血小板血浆来源外泌体的作用包括抑制软骨细胞凋亡,促进软骨细胞增殖与迁徙,抑制炎症因子分泌,促进细胞外基质合成与分泌;③在机制方面,涉及PI3K/Akt、Erk1/2、Akt/Bad/Bcl-2及Wnt/β-连环蛋白信号通路。

低温、高pH胁迫对水稻幼苗根系质膜、液泡膜ATP酶活性的影响

以耐冷性不同的两个水稻品种为材料 ,比较研究了幼苗根系质膜、液泡膜ATP酶对低温 (8℃ )及高pH (8.0 )胁迫的反应。结果表明 :水稻根细胞质膜和液泡膜上均存在Ca2 + ATP酶 ,但活性远低于H+ ATP酶。耐冷品种武育粳 3号经低温 (8℃ )处理 2d ,根系质膜和液泡膜H+ ATP酶、Ca2 + ATP酶活性均明显升高 ,至冷处理 12d ,H+ ATP酶、Ca2 + ATP酶活性有所下降 ,但仍与对照相近 ;而冷敏感品种汕优6 3经低温 (8℃ )处理 2d ,根系质膜H+ ATP酶活性略有升高 ,而质膜Ca2 + ATP酶以及液泡膜H+ ATP酶、Ca2 + ATP酶活性已明显下降 ;至冷处理 12d ,4种酶活性均明显低于对照。高 pH胁迫使质膜和液泡膜H+ ATP酶活性下降 ,而使Ca2 + ATP酶活性上升。高 pH胁迫会加剧低温冷害。结果表明 ,耐冷品种质膜。

大豆下胚轴质膜H^+-ATPase质子转运的测定

以大豆下胚轴为材料，采用改进的匀浆介质，通过两相法制得具有质子转运活力的高纯度质膜微囊．并且发现冻融处理可以促进质膜微囊的翻转而提高荧光猝灭效率．质子载体和质子转运特性分析表明，由Ｍｇ２＋ＡＴＰ引发的荧光猝灭可以被质子载体ＣＣＣＰ恢复，并被质子通道抑制剂ＤＣＣＤ抑制；并且发现质膜Ｈ＋ＡＴＰａｓｅ专一抑制剂钒酸钠可以完全抑制荧光猝灭，同时发现荧光猝灭依赖于Ｍｇ２＋，并受Ｋ＋刺激，最适ｐＨ为６５．以上证明所测荧光猝灭是由质膜Ｈ＋ＡＴＰａｓｅ所进行的质子转运引起的．结果同时表明，维持Ｈ＋ＡＴＰａｓｅ合适构象和提高质膜微囊封闭性是制备具有Ｈ＋转运活力质膜微囊的两个关键因素．

NaCl处理下大麦根系质膜微囊结合多胺与Na^+/H^+逆向运输的关系

NaCl10 0mmol/L处理结合外施Spd和Put以及多胺代谢抑制剂邻二氮杂菲和MGBG ,以改变大麦根系质膜结合多胺种类和数量 ,研究了大麦根系质膜上两种形态多胺与质子泵和Na+ /H+ 逆向运输活性的关系。结果发现 ,NaCl处理后大麦根系质膜微囊上存在Na+ /H+ 逆向运输活性。质膜H+ ATPase活性与膜上非共价键结合多胺数量间呈显著正相关 ,其中 ,Spd对H+ ATPase的激活程度大于Put。膜蛋白上共价键结合多胺数量与Na+ /H+ 逆向运输活性间呈极显著正相关关系 ,说明大麦根系质膜Na+ /H+ 逆向运输的盐诱导似乎与Na+ /H+ 逆向运输蛋白的从头合成有关。此外 ,质膜Na+ /H+ 逆向运输活性仅与膜蛋白上共价键结合多胺数量有关 ,而与多胺种类关系不大。

经渗透胁迫高梁根的质膜囊泡K^＋—ATP酶及其运输特征

经ＰＥＧ－１０００处理的高粱根，用不连续蔗糖密度梯度制备获得反转密闭的纯化质膜囊泡，显示一种特殊的ＡＴＰ酶活力，它不同于Ｈ＋－ＡＴＰ酶，其最适ＰＨ为７．５，具有高ＡＴＰ亲和力和较低的Ｋ＋转运能力。环己酰亚胺和钒酸钠能抑制其活力，表明它是新合成的Ｐ－型ＡＴＰ酶。

不同生境下木麻黄脯氨酸含量和质膜ATPase活性的研究(英文)

采集生长于恶劣环境和中生环境的普通木麻黄(Casuarina)小枝,超速离心提取粗质膜制剂后,用两相系统法纯化得到质膜微囊,研究不同生境下木麻黄的质膜ATPase活性,并测定木麻黄小枝的游离脯氨酸含量。实验结果表明:同一生境中的木麻黄ATPase活性相对一致,而同一树种木麻黄不同生境下质膜微囊H^+-ATPase、Ca^(2+)-ATPase、K^+-ATPase活性有显著差异,表现出以渗透胁迫为主的恶劣环境下的木麻黄质膜微囊ATPase活性和木麻黄细胞内游离脯氨酸明显高于中生环境下生长的木麻黄。说明普通木麻黄在干旱和盐胁迫下能调整生理生化过程来提高其质膜ATPase活性和增加细胞内脯氨酸含量提高渗透调节能力以保证其在恶劣环境的正常生长。

ABA,IAA和CaM对发育菜豆子叶质膜H^＋—ATPase活力的效应

以亲水性两相分配法从发育菜豆子叶制备的质膜制剂经冻融循环操作，部分膜微囊可转变成密闭的翻转型。取冻融４次的质膜微囊用于Ｈ＋－ＡＴＰａｓｅ试验表明，ＡＴＰａｓｅ活力为ＡＢＡ和ＣａＭ显著地激活，但受ＩＡＡ显著抑制；质子泵活力被ＡＢＡ显著促进，但为ＣａＭ显著抑制，ＩＡＡ对质子泵活力无显著效应。可以认为：ＡＢＡ促进发育菜豆子叶吸收光合同化物可能是通过促进质膜Ｈ＋－ＡＴＰａｓｅ活力，从而促进质子／蔗糖同向运输而获得；ＩＡＡ则可能对菜豆子叶的质膜Ｈ＋－ＡＴＰａｓｅ无显著效应。在激素信号传导途径中，ＣａＭ对质膜Ｈ＋－ＡＴＰａｓｅ活力可能无直接影响。

小麦根质膜H^+-ATPase的部分纯化

以小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）根为材料，采用不连续蔗糖密度梯度离心法制备高纯度质膜微囊。质膜经ＴｒｉｔｏｎＸ１００和ＫＣｌ处理后，再用Ｚｗｉｔｅｒｇｅｎｔ３１４增溶Ｈ＋ＡＴＰａｓｅ，最后用硫酸铵沉淀得到部分纯化的质膜Ｈ＋ＡＴＰａｓｅ。ＳＤＳＰＡＧＥ结果表明，经过上述步骤纯化，分子量为９４ｋＤ的膜蛋白组分得到富集；与质膜相比，其含量提高１５．７倍。部分纯化的质膜Ｈ＋ＡＴＰａｓｅ可以水解ＡＴＰ，受Ｋ＋刺激，并被Ｎ，Ｎ′ｄｉｃｙｃｌｏｈｅｘｙｌｃａｒｂｏｄｉｍｉｄｅ（ＤＣＣＤ）抑制；ＡＴＰ水解活力被Ｎａ３ＶＯ４抑制９５％，但不被ＮａＮ３、ＮａＮＯ３和Ｎａ２ＭｏＯ４抑制。

精浆胞外囊泡对精子功能的作用研究进展

近年来,体液中的胞外囊泡受到特别关注,许多物种精浆中均含有胞外囊泡,这些精浆胞外囊泡可以影响精子功能,还可作为雄性动物繁殖力或人类不孕不育相关疾病的标志。本文综述了精浆胞外囊泡对精子功能作用的研究进展,以期更好地理解精子受精能力的调控机制,同时为畜牧生产中提高雄性动物繁殖力和男性生育相关疾病治疗提供一定的思路和参考。

侵染细胞质膜附近小泡的来源及其作用

箭舌豌豆根瘤幼龄侵染细胞的壁和质膜比较光滑，成熟侵染细胞与此不同，不仅细胞壁厚薄不均，有较多的胞间连丝，而且质膜常常内陷形成各种突起，然后脱离质膜形成小泡。这些位于质膜附近的小泡体积较小，多呈圆形，既可单独存在，也可多个聚在一起。在向细胞中央移动中，有的小泡靠近细胞质膜，甚至与细菌周膜融合。有的小泡还可与附近的小液泡融合变为较大液泡，并常有降解程度不同的细菌位于其中。在衰老侵染细胞中，细胞壁附近有小泡。

玉米根细胞正面向外和内翻外质膜囊泡的分离

利用水双相分配分离法从玉米根细胞提取原生质膜囊泡。其质膜成分特异的K^+,Mg^(2+)—ATP酶活性比膜粗提液高1.67倍。其他细胞器标记酶活性在质膜组分中很低或没有。特异染色电镜形态学定量统计表明质膜组分中90％以上是质膜囊泡。去污剂刺激ATP酶活性实验证明,约92％囊泡是正面向外的。应用此法也证明NAD(P)H—氧化酶可能是跨膜存在的,其催化Fe^(3+)还原的位点可能在膜的内侧。应用高盐冻—融法并结合水双相分配分离法处理正面向外囊泡,可获得内翻外质膜囊泡,实验证明它不仅纯度高而且封闭。