Adenosine, Inosine, and Guanosine Protect Glial Cells During Glucose Deprivation and Mitochondrial Inhibition: Correlation Between Protection and ATP Preservation

Abstract: The purpose of this study was to determine the mechanism by which adenosine, inosine, and guanosine delay cell death in glial cells (ROC-1) that are subjected to g lucose d eprivation and m itochondrial respiratory chain inhibition with amobarbital (GDMI). ROC-1 cells are hybrid cells formed by fusion of a rat oligodendrocyte and a rat C6 glioma cell. Under GDMI, ATP was depleted rapidly from ROC-1 cells, followed on a much larger time scale by a loss of cell viability. Restoration of ATP synthesis during this interlude between ATP depletion and cell death prevented further loss of viability. Moreover, the addition of adenosine, inosine, or guanosine immediately before the amobarbital retarded the decline in ATP and preserved cell viability. The protective effects on ATP and viability were dependent on nucleoside concentration between 50 and 1,500 µ M . Furthermore, protection required nucleoside transport into the cell and the continued presence of nucleoside during GDMI. A significant positive correlation between ATP content at 16 min and cell viability at 350 min after the onset of GDMI was established ( r = 0.98). Modest increases in cellular lactate levels were observed during GDMI (1.2 nmol/mg/min lactate produced); however, incubation with 1,500 µ M inosine or guanosine increased lactate accumulation sixfold. The protective effects of inosine and guanosine on cell viability and ATP were >90% blocked after treatment with 50 µ M BCX-34, a nucleoside phosphorylase inhibitor. Accordingly, lactate levels also were lower in BCX-34-treated cells incubated with inosine or guanosine. We conclude that under GDMI, the ribose moiety of inosine and guanosine is converted to phosphorylated glycolytic intermediates via the pentose phosphate pathway, and its subsequent catabolism in glycolysis provides the ATP necessary for maintaining plasmalemmal integrity.     

聚乙二醇丙烯酸酯的合成与表征

以聚乙二醇-400（PEG400）与丙烯酸直接缩合反应,在不加有毒带水剂的条件下合成了丙烯酸聚乙二醇酯（PEGA）。通过正交实验确定酯化反应的最佳条件：丙烯酸／PEG400的摩尔比为2．0：1．0,反应温度是110℃,阻聚剂对苯二酚为0．4％（以醇酸总质量计）,反应时间为6小时,催化剂对甲苯磺酸为0．8％（以醇酸总质量计）,产率为76．7％。产品结构经IR和1HNMR表征,证明是所需的产物。     

Polyethylene Glycol Induced Fusion of Selected Pairs of Single Protoplasts

A new method for polyethylene glycol (PEG) -induced fusion between single pairs of selected protoplasts was developed. The protoplasts were prepared from tobacco leaves. Under an inverted microscope two defined protoplasts were selected with a hand-made micropipette and transferred into a droplet of fusion solution containing 25 % PEG (M. W. 6000), 0. 1 mol/L mannitol and 0. 01 mol/L CaCl2 · 2H2O (pH 5.6). Slightly moving the pipette caused the protoplasts to contact and adhere to each other, the fusion pairs were then transferred to a solution containing 10% PEG, 0.35 mol/L sucrose and 0. 01 mol/L CaCl2 · 2H2O (pH 5.6) for approximately 10 min, followed by subsequent washing with a solution containing 0.45 mol/L sucrose and 0.04 mol/L CaC12 · 2H20 (pH 7—9). Compared with conventional fusion methods adopted to protoplast population, the present method can avoid either blind fusion of protoplasts belonging to one partner and fusion among multiple protoplasts, or the presence of unfused protoplasts, thus ensure the fusion to be precisely at the level of a selected pair of single protoplasts. Moreover, it is simple and convenient enough to show its potentiality for wide application in somatic hybridization and particularly in the case of small quantity of parental protoplasts such as in vitro intergametic fusion studies.     

Cell Swelling, Blebbing, and Death Are Dependent on ATP Depletion and Independent of Calcium During Chemical Hypoxia in a Glial Cell Line (ROC-1)

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.     

Modification of Lipase by Polyethylene Glycol

Lipase was modified using polyethylene glycol activated by p-nitrochloroformate. The hydrolytic activity of the polyethylene glycol-derivatised lipase (PEG-lipase) was relatively low compared with that of the unmodified enzyme in aqueous system. The esterification activity, however, was enhanced following the modification. The rate of esterification of butyric acid was higher than that of oleic acid. Benzene was the best solvent for the esterification reaction.     

Depletion and recovery of ATP in V79 cells with exposure to inhibitors of glycolysis and oxidative phosphorylation

Summary Cells of the cultured hamster cell line V79 were labeled with tritiated adenosine and incubated for up to 30 min in the presence of inhibitors of glycolysis and oxidative phosphorylation. These inhibitors were (a) 5 mM KCN plus 5 mM iodoacetate, (b) 5 mM KCN plus 5 mM KF, and (c) 15 mM KCN plus 15 mM KF. The fate of the tritium label was examined during incubation with inhibitors and also during subsequent incubation in growth medium in the absence of inhibitors. The tritiated ATP pool was found to decrease in cells incubated in the presence of any of the inhibitor combinations, but only in the presence of 15 mM KCN plus 15 mM KF was this pool decreased below the level of detection. After cells were incubated with KCN plus KF, a high level of ATP was recovered when the inhibitors were removed. Cells incubated with KCN plus iodoacetate retained depletion levels of ATP. Plating efficiency and trypan blue staining showed that KCN-KF treated cells retained viability, whereas KCN-iodoacetate treated cells did not. Cells were examined for ability to take up tritiated uridine before, during, and after depletion of ATP by incubation in the presence of 15 mM KCN plus 15 mM KF. These cells were found to have a variation in uridine uptake that was related directly to intracellular ATP level. Cells in which the ATP was very low exhibited little or no uridine uptake, whereas cells in which the ATP level was near normal exhibited normal uridine uptake. This work was supported in part by Grant GM24271 from the National Institutes of Health, Bethesda, Maryland.     

黄蝉离体生殖细胞的融合

近期研究者用不同的方法成功地从花粉和花粉管中分离出生殖细胞(Zhou等1986,1988;Zhou 1988,Tanaka 1988,Tanaka等1989,周嫦和吴新莉1990,徐是雄1991a)。这些生殖细胞在离体培养的条件下,不但能够保持生活力,而且还能     

Studies Upon the Toxicity of Polyethylene Glycol Coated Lipid Vesicles: Acute Hemodynamic Effects,Pyrogenicity and Complement Activation

Abstract

The toxicity of PEG-coated liposomes has been examined by testing the ability of such vesicles to (a) initiate a fever response, (b) activate complement and (c) alter various hemodynamic parameters in rabbits. The results indicate that DSPC/Chol/DMPG/Vit.E/ PE-PEG (45.5:40:9:1:4.5) vesicles injected I.V. in rabbits at a lipid dose of ～4 μmole/kg do not elicit a fever response either due to the presence of bacterial contamination, or by activating the release of endogenous pyrogens and, in addition, cause no statistically significant (Mann-Whitney p >0.05) changes in various hemodynamic parameters compared to injection of saline. An in-vitro hemolytic assay indicated that these same vesicles cause no activation of complement over the lipid concentration range 0.06–64 mM. These results are discussed in terms of the potential use of PEG-coated vesicles as blood pool imaging agents in nuclear medicine.     

用聚乙二醇分离富集microRNA的方法探讨

目的探讨用聚乙二醇（polyethylene glycol,PEG）溶液分离富集miRNA的操作方法和分离富集效果,并与Ambion公司的miRNA分离试剂盒分离效果进行比较。方法用PEG溶液和Ambion公司的miRNA分离试剂盒从肝脏组织总RNA中分离富集miRNA,用变性琼脂糖和变性聚丙烯酰胺凝胶电泳鉴定分离效果,并在富集的miRNA中用RT—PCR扩增miR-122以鉴定是否有效地回收了miRNA。结果PEG和Ambion公司的miRNA分离试剂盒都能有效地富集miRNA,PEG富集的RNA片段比Ambion公司的试剂盒的大。结论PEG溶液能有效地分离富集miRNA,和Ambion公司的miRNA分离试剂盒分离效果相当,并具有操作简便、快捷及成本低廉的优点。     

A Phase-Transfer Glucosamination of Phenols Catalyzed by Polyethylene Glycol

Glycosylation of phenols with α-D-glucosaminyl chloride peracetate catalyzed by polyethylene glycol (PEG) was carried out in a solid-liquid phase transfer system at room temperature. The results were compared with those previously obtained for the catalysis with various crown ethers. The catalytic activity of PEG in this reaction was found to be comparable with those of 15-crown-5 and aromatic crown ethers.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 335–336.Original Russian Text Copyright © 2005 by Kur’yanov, Priskoka, Chupakhina, Chirva.     

Chemical Hypoxia-Induced Cell Death in Human Glioma Cells: Role of Reactive Oxygen Species,ATP Depletion,Mitochondrial Damage and Ca2+

This study was undertaken to evaluate whether chemical hypoxia-induced cell injury is a result of reactive oxygen species (ROS) generation, ATP depletion, mitochondrial permeability transition, and an increase in intracellular Ca²⁺, in A172 cells, a human glioma cell line. Chemical hypoxia was induced by incubating cells with antimycin A, an inhibitor of mitochondrial electron transport, in a glucose-free medium. Exposure of cells to chemical hypoxia resulted in cell death, ROS generation, ATP depletion, and mitochondrial permeability transition. The H₂O₂ scavenger pyruvate prevented cell death, ROS generation, and mitochondrial permeability transition induced by chemical hypoxia. In contrast, changes mediated by chemical hypoxia were not affected by hydroxyl radical scavengers. Antioxidants did not affect cell death and ATP depletion induced by chemical hypoxia, although they prevented ROS production and mitochondrial permeability transition induced by chemical hypoxia. Chemical hypoxia did not increase lipid peroxidation even when antimycin A was increased to 50 M, whereas the oxidant t-butylhydroperoxide caused a significant increase in lipid peroxidation, at a concentration that is less effective than chemical hypoxia in inducing cell death. Fructose protected against cell death and mitochondrial permeability transition induced by chemical hypoxia. However, ROS generation and ATP depletion were not prevented by fructose. Chemical hypoxia caused the early increase in intracellular Ca²⁺. The cell death and ROS generation induced by chemical hypoxia were altered by modulation of intracellular Ca²⁺ concentration with ruthenium red, TMB-8, and BAPTA/AM. However, mitochondrial permeability transition was not affected by these compounds. These results indicate that chemical hypoxia causes cell death, which may be, in part, mediated by H₂O₂ generation via a lipid peroxidation-independent mechanism and elevated intracellular Ca²⁺. In addition, these data suggest that chemical hypoxia-induced cell death is not associated directly with ATP depletion and mitochondrial permeability transition.     

聚乙二醇二缩水甘油醚交联氨基载体LX-1000EA固定化脂肪酶 *

聚乙二醇二缩水甘油醚(PEGDGE)作为双功能环氧试剂,在实验中被用于交联氨基载体LX-1000EA共价固定化海洋脂肪酶,经过处理后的载体共价固定化脂肪酶具有良好的效果。实验经过单因素初筛和正交试验,得到最佳的交联及固定化条件为0.75%交联剂浓度、交联温度35℃、交联时间3h、载体量1.25g、pH9.0、固定化温度55℃、固定化时间1h。对LX-1000EA-PEGDGE固定化酶与游离酶、戊二醛(GA)交联LX-1000HA-GA的固定化酶进行酶学性质的比较,发现LX-1000EA- PEGDGE固定化酶较游离酶最适反应温度未改变,与LX-1000HA-GA相同的是最适反应pH都由7.0提高为8.0。在最适条件中所测LX-1000EA-PEGDGE酶活达到78.84U/g,固定化改变了游离酶的酸碱耐受性,热稳定性和操作稳定性较游离酶和LX-1000HA-GA固定化酶均有提高。LX-1000EA-PEGDGE的热稳定表现优异,在60℃孵育3h后保留90%酶活;使用5次后仍能残余50%酶活;保存30天酶活仍保留60%。首次使用新型双环氧交联剂PEGDGE交联有机氨基载体共价结合固定化脂肪酶,为更有效的固定化方法提供了技术支持,同时也发现交联剂对固定化酶的性质存在较大影响。     

固定化细胞法在5'-三磷酸腺苷合成中的应用

从生物催化活性细胞、细胞的固定化形式、固定化细胞反应器3个方面对固定化细胞法合成5’-三磷酸腺苷(ATP)的研究情况作了扼要综述。分析了我国ATP生产的工业化现状,并对今后的研究和开发提出了建议。     

乳果糖口服液联合聚乙二醇电解质散剂在便秘患者行结肠镜检查前肠道准备中的应用研究

目的:探讨便秘患者行结肠镜检查前联合应用乳果糖口服液联合聚乙二醇电解质散剂的临床效果。方法:将90例接受结肠镜检查的便秘患者分为实验组和对照组,每组各45例,实验组肠镜检查前1天口服乳果糖口服液及聚乙二醇电解质散剂,对照组检查前1天口服聚乙二醇电解质散剂,比较两组患者的首次大便时间(开始服药至初次大便时间)、排便次数、大便清澈时间(初次大便至大便清澈时间)、肠道准备清洁度和不良反应情况。结果:实验组首次大便时间、大便清澈时间均短于对照组,差异有统计学意义(P0.05)。实验组完成肠道准备的排便次数较对照组次数增多,差异有统计学意义(P0.05)。实验组肠道准备清洁度优于对照组,差异有统计学意义(P0.05)。两组不良反应发生率无明显差异(P0.05)。结论:便秘患者结肠镜检查前肠道准备中联合应用乳果糖口服液联合聚乙二醇电解质散剂可提高肠道清洁度,增加排便次数,缩短大便清澈时间,达到理想的清肠效果,有助于发现微小病变,降低肠镜检查的漏诊率。     

Highly Efficient Aqueous‐Processed Hybrid Solar Cells: Control Depletion Region and Improve Carrier Extraction

Environmental friendly aqueous‐processed solar cells have become one of the most promising candidates for the next‐generation photovoltaic devices. Researchers have made lots of progress in designing active materials with novel structures, manipulating the defects in active materials, optimizing device architecture, etc. However, it has long been a challenge to control the width of the depletion region and enhance carrier extraction ability. Fabrication of a thick bulk heterojunction (BHJ) film is an effective strategy to address these issues but difficult to realize. Herein, the thicker BHJ film of ZnO:CdTe is successfully fabricated and incorporated into CdTe‐poly(p‐phenylenevinylene) hybrid solar cells. As expected, this BHJ film enhances light absorption, extends the width of the depletion region, prolongs carrier lifetime, and promotes carrier extraction ability. Moreover, the electron transport layer of sol–gel ZnO with excellent transmittance and electrical conductivity boosts electron generation, transport, and injection, which further improves the device performance. As a result, the highest short current density (J_sc) of 19.5 mA cm^?2, power conversion efficiency of 6.51%, and the widest depletion region (177 nm) are obtained in aqueous‐processed hybrid solar cells.     

聚乙二醇重组人粒细胞刺激因子Ib期临床NSCLC人体试验的安全性研究

目的:考察非小细胞肺癌患者化疗后对HHPG-19K的耐受性及该药物的安全性.方法:随机将招募受试者30例平均分成设五个试验组:HHPG-19K 3个剂量组(60 μg/Kg、100 μg/Kg、200 μg/Kg)、阳性对照组(惠尔血,即G-CSF 5 μg/Kg/天)和阴性对照组,对比5组的安全性观察指标.结果:3个剂量组均出现不良事件,占总人数的100％;另外,实验室检查值的异常主要有ALP、ALT及AST升高等.不良事件的类型和严重程度均为轻中度,与阳性对照组无差异(P＜0.05).而阴性对照组发生不良事件为13％,少于剂量组与阳性对照组(P＜0.05).结论:试验用药物HHPG-19K在非小细胞肺癌化疗患者中具有较好的耐受性,且不产生耐药性抗体,其中100 μg/kg安全性更好.     

聚乙二醇沉淀剂有效分离尿外泌体

尿外泌体是病毒大小的胞外囊泡,是非侵入性获得肾及泌尿生殖道细胞生理病理信息的重要靶标。聚乙二醇沉淀 剂可经济高效地分离富集血清等外泌体,但未见用于尿外泌体富集的详细报道。本研究采用聚乙二醇沉淀剂分离鉴定尿外泌体,并对其RNA组分进行检测,以期建立一个经济、高效、简便的尿外泌体分离富集方法。采集10例健康志愿者晨尿20 mL,聚乙二醇沉淀剂分离尿外泌体。透射电镜观察到直径30～100 nm双层膜包绕的囊性小泡,中央有直径5～15 nm高电子密度区。Western印迹检测到外泌体标记蛋白CD63、CD9、TSG101、ADAM10和内标蛋白β-肌动蛋白的表达。纳米粒径仪测定粒子直径介于30～130 nm,并可见25.37 nm和95.07 nm二个粒子峰。qRT-PCR扩增得到β-肌动蛋白和RNU6 RNA产物带。上述结果表明,聚乙二醇沉淀剂可分离富集尿外泌体,该法简单、高效,不需要超速离心机等昂贵设备,且采用该法富集到的外泌体可用于后续蛋白质与核酸分析。该方法可望加速液体活检应用,尤其是肾及泌尿生殖道病变的无创检测。     

Manganese-Induced Neurotoxicity is Differentially Enhanced by Glutathione Depletion in Astrocytoma and Neuroblastoma Cells

Manganese (Mn) is neurotoxic: the underlying mechanisms have not been fully elucidated. l-Buthionine-(S,R)-sulfoximine (BSO) is an irreversible inhibitor of γ-glutamylcysteine synthetase, an important enzyme in glutathione (GSH) synthesis. To test the hypothesis that BSO modulates Mn toxicity, we investigated the effects of treatment of U-87 or SK-N-SH cells with MnCl₂, BSO, or MnCl₂ plus BSO. We monitored cell viability using MTT assay, staining with HO-33342 to assess live and/or apoptotic cells, and staining with propidium iodide (PI) to assess necrotic cells; we also measured cellular glutathione. Our results indicate decreased viability in both cell types when treated with MnCl₂ or BSO: Mn was more toxic to SK-N-SH cells, whereas BSO was more toxic to U-87 cells. Because BSO treatment accentuated Mn toxicity in both cell lines, GSH may act to combat Mn toxicity. Thus, further investigation in oxidative stress mediated by glutathione depletion will unravel new Mn toxicity mechanism(s).